It is noteworthy that in the sensitivity analysis is the adjusted HR in the group with depression diagnosis was 3.392 and in the oral cancer subgroup of “Tongue” was 2.539. This study shows oral cancer was associated with a significantly increased risk for developing subsequent depression and early identification and treatment of depression in oral cancer patients is crucial.This study investigates an association between oral cancers and the risk of developing depression. We conducted a total of 3031 patients with newly diagnosed oral cancers and 9093 age-, sex-, and index year-matched controls (1:3) from 2000 to 2013 were selected from the National Health Insurance Research Database (NHIRD) of Taiwan. After adjusting for confounding factors, multivariate Cox proportional hazards analysis was used to compare the risk of depression over a 13-year follow-up. Of the patients with oral cancer, 69  developed depression compared to 150  in the control group. The Cox proportional hazards regression analysis showed that the adjustment hazard ratio (HR) for subsequent depression in patients with oral cancer diagnosed was 2.224 (95% Confidence Interval [CI] 1.641–3.013,  Almost 50% of head and neck cancers arise in the oral cavity, there were an estimated 355,000 new cases and 177,000 deaths worldwide for oral cavity cancer in 20182. Furthermore, there are approximately 4100 newly diagnosed oral cavity cancer cases in Taiwan annually, which ranks the 4th most common cancer in males and the leading cause of cancer death in males4. More than half of patients diagnosed with oral cavity cancer are in stage III or IV of the disease4. This requires major treatments such as radical excision, radiation therapy (RT), and surgery with concurrent chemoradiation therapy (CCRT)5. These treatments can cause severe physical and functional impairments that are also associated with the development of depression7. Changes in overall symptom severity, including swallowing difficulties, poor appetite, oral mucositis, pain, fatigue, and a dysmorphic appearance were all found to be significantly related to the severity of oral-cancer patients’ depression. Psychological problems require careful monitoring and special attention after treatment8.Head and neck cancers are the 7th most common cancer, the 5th most common in men and the 12th most common in women worldwide9. Depression has been found to be related to cancer patient symptom severity and level of physical functioning11. Lower survival rates and poorer outcomes have been demonstrated in cancer patients with depression14. Recent evidence pointed out that depression can be seen as an independent factor for cancer survival15. The need for comprehensive assessments and routine symptom evaluation in management plans are emphasized because of the significant prevalence and severity of depression among new and follow-up cancer patients16. Overall, depression adversely affected patient quality of life and may also interfere with treatments and rehabilitation18.Depression is a major public health problem and has an especially large impact on physical health when it appears as a comorbidity to a chronic medical condition20. Up to 2021, only one systemic review21 study that link oral cavity cancer as a risk factor for depression with varied severity of symptoms after cancer treatment. Among the previous studies, Jansen et al. (2018)22 and Kuma et al. (2018)23, using validated questionnaires, found that oral cancer is associated with the risk of depression. In addition, Rana et al. (2015)24 have compared the risk of depression in the patients with oral squamous cell carcinoma and oral lichen planus.Previous studies have reported an association between depression and survival in patients with head and neck (HN) cancerWe hypothesize that oral cancer is associated with an increased risk of subsequent depression. Therefore, we conducted this study to identify the association between oral cancer and depression and the potential effect modifiers, by using data from a nationwide health insurance database, the Taiwan National Health Insurance Research Database (NHIRD).Figure p < 0.001). However, the difference between oral cancer group and non-oral cancer group does not achieve significant (log-rank test p = 0.199).Figure p < 0.001), stroke (p < 0.001), and anxiety (p = 0.004).Table p < 0.001; C2 p = 0.001), hyperlipidemia, HTN, COPD, and renal disease (p < 0.001); and more lived in the highest urbanized areas, southern areas of Taiwan (p < 0.001), and more receive treatment in a hospital center with higher medical costs (NT$) and had longer length of days (p < 0.001).Compared to both controls (non-cancer (C1) and non-oral cancer(C2)), the study subjects tended to have lower rates of DM (C1 p < 0.001). After adjusting for age, gender, comorbidities, geographical area of residence, urbanization level of residence, and monthly income, the adjusted HR was 2.224 .Table p < 0.001), 5.978 (p < 0.0013), and 3.109 (p < 0.001), respectively. The patients with DM and renal disease tended to have a lower risk for developing depression before adjustment; however, this became insignificant after adjustment.In oral cancer vs. non-cancer group, the adjusted HR’s for the patients with alcohol abuse, anxiety, and sleep disorder for developing depression were 10.631 (p = 0.047) and received treatment in hospital centers (0.573 (p = 0.001)) and regional hospitals (0.697 (p = 0.025)) tended to have a lower risk for developing depression than those who visited a local hospital. An increase in stay by 1 day also increases the risks for developing depression by 1.9% (p < 0.001).The male patients adjusted HR was 0.687 (Table Table p = 0.001), tongue 2.539 (p < 0.001), gums 2.163 (p = 0.020), floor of mouth 2.791 (p = 0.031), Cheek mucosa 1.872 (p = 0.029), and Others 2.326 (p < 0.001).Table p < 0.001), ≧1 year, < 3 years 2.604 (p < 0.001), ≧3 year, < 5 years 1.498 (p = 0.001), and ≧5 year 1.364 (p = 0.013) were associated with a higher risk developing depression and hazard ratio show decline tendency as follow-up time was longer.Table This study showed that patients with a diagnosis of oral cancer has nearly 2.2-fold increased risk of developing depression. Kaplan–Meier analysis revealed that the study subjects had a significantly lower 13-year depression-free survival rate than the controls. In addition, it took 1 year to achieve a significantly adjusted HR, and therefore, the duration of 13 years appears to be a reasonable period to follow patients with newly diagnosed oral cancer. To the best of our knowledge, this is the first population-based study on the topic of the increased risk of depression in the patients with oral cancer.25. Another previous study on Taiwan focused on early post-surgery stage patients with oral cancer and showed that 18.2% met the definition of depression26. A study about the Taiwan NHIRD further indicated that newly diagnosed oral cavity cancer patients age >  = 50 had a significantly lower 5-year depression event-free survival rate with 2.53-fold hazard ratios higher than controls27.A study done in Hong Kong concluded that approximately 8% of the oral cancer study participants met the clinical cut-off for depression28.Our study results show a 2.224-fold greater risk than controls that had a 2.28% oral cancer diagnosed with depression. Further, our study indicated a greater number of patients, broader age groups, and a longer follow-up period. More detailed data about each subgroup according to ICD9 area and sensitivity analysis was collected and provided. A low prevalence of depression was reported in this study, which might be rooted in the cultural context. Taiwan male adults tend to demonstrate a kind of stoicism that may be reflected in a much lower percentage in help-seeking behaviors30.In this study, a patient diagnosed with oral cancer in the lip, tongue, and other cancer subgroups show a higher significant hazard ratio than others Table . The oth31. Patients who underwent 3/4 or total anterior glossectomy had poorer functional outcomes than those who underwent either 1/4 or 1/2 glossectomy32. A previous study showed that depression and swallowing function were highly correlated33. Patients with tumors of the tongue had worse functional dysphagia than those with cancers in other areas of the oral cavity34. Dysphagia contribute to malnutrition, dehydration, weight loss, reduced functional abilities, and fear of eating and drinking may also lead to depression and reduced quality of life35. Early identification and management of dysphagia can improve treatment outcomes and reduces the development of depression31.The tongue and soft palate are the most important organs in the oral cavity and oropharynx for speech and swallowing33. A recent study showed that dysfunction in salivation, problems with eating, and problems with social contact were major risk factors for the development of depression36.The direction of the relationship among depression, swallowing function, and quality of life for HNC patient remains unclear29.In Taiwan, the majority of oral cavity cancer patients belonged to a lower economic group and had a long history of smoking, chewing betel nut, and drinking alcohol37, which were also correlated with depression40. A synergistic effect on depression might develop from the sudden cessation of the afore-mentioned substance used after receiving the cancer diagnoses26.Oral cancer represent a psychosocial challenge for patients, their families, and the diagnosing provider41. Financial struggle causes significant stress in patients undergoing oral cancer treatment42. Oral cancer survivors do not always return to work after rehabilitation43. Patients may encounter several physical challenges that may influence patient employability and an ability to maintain or continue employment44.Our study indicated that most oral cancer patients come from a lower annual income backgrounds, which may contribute to increasing the hazards for subsequent depression. A previous study showed a large percentage of patients who had received oral cancer treatment indicated a significant financial impact , especially for patients with limited financial resources46. During treatment, a higher consumption of painkillers, especially those with narcotic analgesics, has been reported to have a higher risk of depression48. Therefore, both oral cancer and its treatment disrupts core aspects of daily life, which may further damage a patient’s self-esteem and encourage social isolation49.Oral cancer surgery often causes significant loss of function, and further radiotherapy and chemotherapy caused side-effects such as nausea, vomiting, mucositis, pharyngitis-related dysphagia, masticatory disorders, and possible loss of complete physiological functions36, which is similar to our study result especially in < 1 year group, i.e. they showed a significant 3.392 hazard ratio , an oral cancer group, a non-oral cancer group, and depression over a 13-year period from the Longitudinal Health Insurance Database (LHID) in Taiwan (2000–2013). The National Health Insurance (NHI) Program was launched in Taiwan in 1995, and as of June 2009 it included contracts with 97% of the medical providers in Taiwan with approximately 23 million beneficiaries, or more than 99% of the entire population in Taiwan65. All diagnoses of depression were made by board-certified psychiatrists and oral cancers were confirmed by oral surgeons or otolaryngologists, and other cancers were confirmed by medical experts who specialized in oncology. The Catastrophic Illness Certificates (CIC) status was used to verify the diagnosis of oral or other cancers. The NHI Administration randomly reviews the records of 1 in 100 ambulatory care visits and 1 in 20 in-patient claims to verify the accuracy of diagnoses66. Several studies have demonstrated the accuracy and validity of diagnoses in the NHIRD69.The NHIRD uses the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) codes to record diagnosesThis study was a retrospective matched-cohort design. Patients with newly diagnosed oral cancer were selected from 1 January 2000 to 31 December 2013 according to ICD-9-CM codes at the sites of lips, tongue, major salivary glands, gums, floor of mouth, cheek mucosa, and other sites in the oral cavity , urbanization level of residence (level 1–4), and monthly income (in New Taiwan Dollars (NTD): < 18,000, 18,000–34,999, and ≥ 35,000). The urbanization level of residence was defined according to the population and various indicators of the level of development. Level 1 was defined as a population > 1,250,000, and a specific designation as political, economic, cultural, and metropolitan development. Level 2 was defined as a population between 500,000 and 1249,999, and as playing an important role in the political system, economy, and culture. Urbanization levels 3 and 4 were defined as a population between 149,999 and 499,999, and < 149,999, respectivelyBaseline comorbidities included diabetes mellitus (DM), hyperlipidemia, hypertension, alcohol abuse, tobacco use, stroke, chronic obstructive pulmonary disease, ischemic heart disease, renal disease, anxiety, and sleep disorders Table .71.All study participants were followed from the index date until the onset of depression , withdrawal from the NHI program, or through to the end of 20132 were used to evaluate the distributions of categorical and continuous variables, respectively. Multivariate Cox proportional hazards regression analysis was used to determine the risk of depression, and the results were present as hazard ratio (HR) with 95% confidence interval (CI). The difference in the risk of depression between the study  and control groups  was estimated using the Kaplan–Meier method with the log-rank test.All analyses were performed using SPSS software version 22 . The student’s t-tests and χ72. Third, the validation of diagnostic codes of cancers, in the NHIRD, has been confirmed, in accordance with the Taiwan National Cancer Registry (NCR) and the Catastrophic Illness Certificates (CIC) status for oral73 or a wide range of most of the cancers74. The accuracy of the diagnostic codes for the depression or oral cancer could provide a relative reliable association between oral cancer and depression. Fourth, the validation by suicide and antidepressant usage and sensitivity analysis also revealed the association between oral cancer and depression. These findings support evidence of the association between oral cancer and the risk of depression.First, we conducted this population-based study, by LHID retrieved from the NHIRD, a large sample size during the duration of 13 years. A long-term observation allowed for more credibility in this study. Second, one recent study has validated the accuracy of the diagnostic code for major depressive disorder in the NHIRD and supporting the utilization of psychiatric diagnoses in claims databasesThere were several limitations to this study: First, since this is a study in a Taiwanese population, it would be limited in the generalization of the results to other countries. Second, in Taiwan, some specialists other than board-certified psychiatrists, such as neurologists or family medicine physicians, might prescribe antidepressants with the diagnosis of depressive disorder. In addition, depression is significantly underdiagnosed via ICD codes and that survey-based screening data is necessary for full screening of depression risk. Third, even though patients with the diagnosis of depression and oral cancer could be identified using the insurance claims data, the data on the severity, stage, and impact on their caregivers and socio-economic status were not available. Fourth, tobacco cessation was not included in the NHIRD, therefore, it was not matched it in this study. In addition, details regarding the cancer therapy and progression were not fully available in the NHIRD. More research may be necessary to clarify the long-term risk of developing depression in patients with oral cancer.This study was conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). The Institutional Review Board of Tri-Service General Hospital approved this study and exempted the need for individual written informed consent (TSGHIRB No: B-109-40).The Institutional Review Board agreed that informed consent could be waived because these databases are encrypted databases.In our study, oral cancer diagnoses were associated with a significant increased risk for the subsequent development of depression. This study serves as a reminder for dentists and physicians that early identification and treatment of depression in oral cancer patients is crucial. Future studies are needed for better treatment strategies for the oral cancer patients to help with and stave off the development of depression.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.


Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis were assessed by the agar diffusion test and live/dead assay. The proliferation of DPSCs on these scaffolds was determined by the MTT assay, DAPI staining, and DNA extraction. Moreover, the antioxidant and anti-inflammation activity of chrysin-loaded scaffolds on inflamed DPSCs was evaluated. Alkaline phosphatase activity and Alizarin Red S Stain tests were done to evaluate the mineralization of DPSCs seeded on these scaffolds. The chrysin-loaded scaffolds reported antimicrobial effects against evaluated bacterial strains. The proliferation of DPSCs seeded on these scaffolds was increased significantly (p < 0.05). The TNFα and DCF levels in inflamed DPSCs showed a significant decrease in the presence of chrysin-loaded scaffolds (p < 0.05). The ALP activity and formation of mineralized nodules of DPSCs on these scaffolds were significantly increased compared with the control group (p < 0.05). These results indicated that chrysin as an ancient therapeutic agent can accelerate the healing and regeneration of damaged pulp tissue, and this active ingredient can be a potential natural substance for regenerative endodontic procedures.The appropriate endodontic material should eliminate the infection and inflammation to provide a situation for regeneration and healing of pulp tissue besides biomineralization. Chrysin is one of the active ingredients of plant flavonoids, which has significant anti-inflammatory and antimicrobial properties. In the present study, this natural substance was evaluated for antioxidant, anti-inflammatory, and mineralization properties on dental pulp stem cells (DPSCs). SEM, FTIR, and TGA tests were used to determine the successful synthesize of chrysin-loaded scaffolds. The antimicrobial effects of the synthesized scaffold against  The dental pulp stem cells (DPSCs) are present in the living dental pulp and replaced injured odontoblasts, which is essential for the formation of reparative dentin after trauma, carious lesions, and other external stimuli , 2. ThesInflammation of the dentin-pulp complex usually occurs due to the infiltration of bacteria via dental caries , 5. LipoFlavonoids are part of the bioactive polyphenol family, which existed in the plant kingdom –15. PropEscherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes [Chrysin  is a natural flavonoid and is considered an active ingredient of honey, passion fruit, Indian trumpet flower, and European propolis –22. Chryytogenes . This chytogenes , 29. Thiytogenes , 31. Amoytogenes , 33.Despite all the mentioned beneficial characteristics of chrysin, poor solubility in water, rapid intestinal and hepatic metabolism, low physicochemical characteristics, and poor stability are the major drawbacks of this substance limiting its application especially in load-bearing areas , 34. To Due to the useful characteristics of chrysin in hard tissue regeneration such as its antimicrobial, anti-inflammatory effects, besides its mineralization potential, this study is aimed at evaluating the effect of this material on DPSCs as a potent regenerative endodontic biomaterial. In the current study, chrysin was loaded on polycaprolactone-gelatin scaffolds. The successful loading of this natural ingredient and toxicity of the fabricated scaffolds for DPSCs were assessed. Moreover, the antimicrobial activity, anti-oxidative, and anti-inflammatory effects of this scaffold were evaluated besides induction of mineralization in DPSCs.2CL2)  and water, respectively. Chrysin  was dissolved in the PCL solution (5% weight ratio). Then, two solutions with a ratio of 1 : 1 were mixed. In order to prevent the creation of a two-phase solution, polyvinyl alcohol (PVA) aqueous was used (1% volume ratio). The poly-ɛ-caprolactone-gelatin solutions without chrysin were mixed too. The mixtures were homogenized and freeze-dried for 48 hours at -20°C. Fourier transform infrared spectroscopy (FTIR) spectra of poly-ɛ-caprolactone-gelatin (PCL/Gel) and PCL/Gel/Chrysin samples in potassium bromide (KBr) tablets were carried out . Interferograms were collected over the range of 500-4000 cm−1 with a minimal resolution of 2 cm−1 and 100 scans. Moreover, thermogravimetric analysis (TGA)  was fulfilled with a range of temperature (from 25°C to 600°C) at a heating rate of with a heating rate of 10°C per minute under nitrogen atmosphere. Morphology of developed scaffolds was showed using MIRA3 FEG-SEM . For this purpose, freeze-dried sample-covered gold was formed in a vacuum by sputter . Then, the images were observed by a KYKY-EM3200 microscope  at an excitation voltage of 26 kV.1.2 g poly-ɛ-caprolactone (PCL)  polymer and gelatin  were dissolved in 12.5 mL dichloromethane , Pseudomonas aeruginosa , Staphylococcus aureus , and Enterococcus faecalis  using an agar well diffusion method. The strains were grown on Mueller Hinton Agar media (Sigma-Aldrich Co.), and 5 mm dimension circular samples (PCL/Gel and PCL/Gel/Chrysin) were placed in the center of plates. Incubation was performed at 37°C for 24 hours to measure the zone of inhibition surrounding each scaffold.The antibacterial activity was done against four different strains including The viability of bacteria was determined using live/dead bacteria kit . Briefly, dentin discs with a radius of 4 to 5 mm and a thickness of 1 mm were prepared. NaOCl (1.5%) and EDTA 17% (pH 7.4) were used to wash the surface of the discs which were finally immersed in distilled water for 20 min. For sterile discs, the autoclave at 121°C was used for 20 minutes. Each of the discs is contaminated in a microtube containing BHI broth with 0.6% of bacterial suspensions. Samples are incubated at 37°C until 21 days at 100% moisture to facilitate bacterial biofilm formation. On day 21, the samples are gently washed with PBS to remove the culture medium and nonadherent bacteria. The specimens were then placed on the 24-well plates, and scaffolding was used to cover the dentin discs containing: PCL/Gel and PCL/Gel/Chrysin. After 7 days of incubation, the scaffolds were removed using 10 mL of distilled water. Samples stained using the Live/Dead Back Light Bacterial Survival Kit were imaged by Citation 5™. The percentage (%) of live bacteria was calculated using ImageJ analysis software  by the ratio green fluorescence (live bacteria) to the total of the fluorescence intensities of red (dead bacteria) and green fluorescence multiple to 100.Dental pulp stem cells were isolated and characterized as described before . BrieflyμL culture medium containing DMEM high glucose (Gibco Co.), 10% FBS (Gibco Co.), and 1X pen/strep (Gibco Co.) were added to each well, and the cell viability after 1, 3, and 5 days was measured by the MTT test. Briefly, 50 μL of 3-  solution (5 mg/mL) was replaced by 50 μL of culture medium in each well and after 4-hour incubation at 37°C and 5% CO2. MTT/medium solution was replaced by 100 μL DMSO. The absorbances of each sample were read at 570 nm by a multiwell plate reader .After 4 passages, 5000 cells of DPSCs were grown on each PCL/Gel or PCL/Gel/Chrysin scaffold placed into a 96-well plate and without scaffold as control. 200 4 cells per well and cultured for 5 days. Before staining with DAPI, the samples washed with PBS (3x) were fixed using 4% paraformaldehyde for 10 min. After that, the samples washed with PBS (3x) were treated with Triton X-100 to improve cell permeability. Finally, the samples washed with PBS (3x) were stained with DAPI (3000 nm) (5 minutes). Samples were imaged via Cytation™ system .Samples, including PCL/Gel with/without chrysin and control, were stained by DAPI   to assess the scaffold's effects on DPSC proliferation. The sterile scaffold was placed on 24-well culture plates. Then, the plates were seeded with DPSCs at a density of 5 × 10DPSCs (cell density: cells per well) were seeded on 24-well plates in PCL/Gel scaffold with/without chrysin and control for 1, 3, and 5 days. The samples were then lysed before DNA extraction with Trizol reagent  (0.3 mL/well). Chloroform (0.2 mL/well) was then used to separate the DNA from the organic and aqueous phases. Following centrifugation, the interphase phase of the samples (the DNA content) was extracted. The samples were suspended in 0.3 mL of 100% ethanol, 1 mL of sodium citrate (0.1 M), and 2 mL of 75% ethanol. The DNA pellet was then treated with NaOH (8 mM) to break down the cell membrane and extract the DNA. The materials were centrifuged at 12000 × g for 10 minutes to eliminate any insoluble debris. Finally, the supernatant was moved to a new tube, and the pH was set using a HEPES buffer. The DNA amount was measured spectrophotometrically using the NanoDrop 2000 instrument.E. coli  to investigate the concentration of TNF α with and without chrysin-loaded scaffolds. LPS of E. coli is a principal component of gram-negative bacteria that activates the immune system. This part of bacterium is used for induction of inflammatory markers in culture medium in in vitro studies in which the researchers aimed to induce inflammation in dental pulp stem cells [α was assessed using ELISA kits .To evaluate the anti-inflammatory activity of fabricated scaffolds, the DPSCs were expanded on scaffolds with and without chrysin. These cells were stimulated with lipopolysaccharides (LPSs) of em cells –44. The ROS levels of DPSCs were evaluated by the DCFDA-Cellular Reactive Oxygen Species (ROS) Detection Assay Kit . The assay uses the cell-permeant reagent 2′,7′-dichlorofluorescein diacetate (DCFDA), a fluorogenic dye that assesses hydroxyl, peroxyl, and other ROS activities within the cells. After that, DCFDA is deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ROS into 2′,7′-dichlorofluorescein (DCF). This component is highly fluorescent that was detected by fluorescence spectroscopy.ALP activity was measured using p-nitrophenol phosphate as the assay substrate based on the manufacturer's instruction . Briefly, 7 days after seeding of DPSCs on PCL/Gel and PCL/Gel/Chrysin scaffolds, the cells were lysed in alkaline lysis buffer. Samples were incubated at 37°C in a solution containing p-nitrophenol phosphate. The presence of p-nitrophenol was measured at an absorbance of 405 nm. The base of the experiment isTotal cellular protein was determined, and the ALP enzyme activity was normalized to total cellular protein.The ARS test was used to measure the calcium deposition on day 21 of culturing DPSC in differentiation medium, as described previously . Brieflyp value < 0.05 was considered statistically significant.All analyses were carried out in triplicate, and data was reported as the mean ± SD. The results were analyzed by one-way ANOVA and Tukey's post hoc tests using Prism software . −1 (C=O stretching), 2925 and 2858 cm−1 (CH2 stretching), 1244 cm−1 (C-O and C-C stretching), and 1048 and 1165 cm−1 (C-O-C stretching). Moreover, the characteristic absorption peaks of gelatin were observed at 1643 cm−1 for C=O stretch of amide I, 1538 cm−1 for N-H bend of amide II, and 1372 cm−1 for amide III. Further peaks of gelatin were detected at 2948 cm−1, which was for aliphatic groups (-CH2 and -CH3), and 3429 cm−1 for O-H stretch, and 1460 cm−1 for -COO-. The spectrum of PCL/Gel/Chrysin offered a little shift in the position of characteristic bands due to the formation of hydrogen bonds between the chrysin and -OH or -NH2 groups in gelatin and PCL.As demonstrated in Thermal gravimetric analysis of PCL/Gel and PCL/Gel/Chrysin was performed to assess the thermal stability of developed scaffolds . As seenThe inner microstructure of the lyophilized PCL/Gel/Chrysin with present and absent DPSCs was imaged by SEM. As shown in Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis strains (p < 0.01).The antimicrobial abilities of PCL/Gel with and without Chrysin were assessed by the agar diffusion method, and the diameters of the zone of inhibition are shown in E. faecalis, S. aureus, and P. aeruginosa bacterial survivability on PCL/Gel and PCL/Gel/Chrysin, with green representing living bacteria and red indicating dead bacteria. According to p < 0.05). In the PCL/Gel/Chrysin groups, the rate of decreased bacterial growth was significantly higher in the E. faecalis group compared to the P. aeruginosa group (p < 0.05).The cell viability of seeded DPSCs after 1, 3, and 5 days was compared with the control group. As demonstrated in p < 0.05). The DNA content recovered from PCL/Gel/Chrysin was about 4-5 times that of the control groups. There is no significant difference in DNA concentration among PCL/Gel/Chrysin samples on different days of culture. Cells grown on PCL/Gel on days 3 and 5 showed a significant increase compared to day 1. However, analyzing the DNA concentration of DPSCs on day 5 revealed that the PCL/Gel/Chrysin group had higher DNA content than the PCL/Gel group , control (DPSCs+LPS), PCL/Gel, and PCL/Gel/Chrysin groups, respectively. The increased levels of this proinflammatory cytokine were reported in the PCL/Gel. However, this increase was not statistically notable. The addition of chrysin in the scaffold remarkably decreased the levels of TNFα.As shown in DCF levels were measured as antioxidant parameters, and the results were demonstrated in p < 0.05).The ALP activity of DPSCs as determined in ∗p < 0.05).The mineralization of DPSCs in these scaffolds is shown in ε-caprolactone) (PCL) is a biodegradable and biocompatible polymer with various applications in regenerative medicine. This biopolymer has superior mechanical strength and could balance the solubility of natural components [The aim of regenerative endodontic is to stimulate the regeneration of the damaged dentin-pulp complex. In dentin-pulp tissue, stimuli such as deep caries lead to differentiation of DPSCs to odontoblasts, which synthesize reparative dentin to preserve pulp vitality. However, the progression in carious lesions could lead to the failure of this defensive process . The actmponents –54. Gelamponents . In the E. faecalis is one of the main resistant species in infected root canals and failed root-filled canals [E. faecalis reduces the expression of Runx2, osterix, and type I collagen genes and interferes with the hard tissue regeneration process [P. aeruginosa is another gram-negative bacteria that was founded in necrotic pulp and pulpitis [E. faecalis in infected root canals [The leakage of bacteria and their by-products is the main cause of infection and inflammation in pulp tissue. The gram-negative bacteria have LPS in their cell walls that play a significant role in the inflammation of pulp tissue. d canals , 57. Thi process , 59. P. pulpitis . A. baumpulpitis . Differepulpitis , 63. Thet canals . Moreovet canals . Chrysint canals , 66. In The proliferative effects of propolis and its active ingredient on adipose-derived stem cells (ADSCs) and Stem Cells from Exfoliated Deciduous Teeth (SHED) were proved in previous studies , 67. In α and DCF levels in chrysin-loaded scaffolds. However, the slight increases in levels of these markers were identified in inflamed DPSCs seeded on polycaprolactone-gelatin scaffolds. PCL is a nontoxic and safe polymer. However, the study conducted by Lee et al. exhibited mild inflammatory responses, which could relate to the accumulation of ROS surrounding the PCL-based scaffold [Hydrogen peroxide, hydroxyl radicals, and superoxide anion are common reactive oxygen species (ROS), which were increased around inflamed cells and tissues in the human body , 69. Thescaffold . TherefoMineralization and formation of the continuous calcified barrier are important for the regeneration of the dentin-pulp complex especially in open apices .During mineralization, calcium deposition occurs on the surface structure that is continued by the accumulation of phosphate ions, which turn the structure into the nucleation and growth sites of calcification. The red stains in ARS are formed due to the bonding of this agent to the calcium ions in calcified ECM . NucleatIn our study, the significant increases in ARS stained nodules were consistent with high ALP levels in the PCL/Gel/Chrysin. These results revealed the capacity of DPSCs to differentiate into various cells, which secrete mineralized matrix in the presence of chrysin. Therefore, these findings provide evidence to prove that chrysin as a natural active ingredient can promote the odontoblastic differentiation of DPSCs besides elimination of infection and inflammation, which make it an appropriate alternative for the regeneration of the dentin-pulp complex.α and DCF. Moreover, the loaded chrysin in a scaffold structure plays a key role as a bioactive agent with the ability to stimulate nucleation sites of mineralization besides enhancement of ALP activity. Thus, it has been concluded that these natural ingredient-based scaffolds could be attractive for the regeneration of damaged dentin-pulp complex.The ability to regenerate dentin-pulp complex with natural components would be a promising alternative in regenerative endodontic procedures. In the current study, the active ingredient of propolis was loaded in polycaprolactone-gelatin scaffolds. These scaffolds showed significant antimicrobial effects against bacterial strains found in the oral cavity. The cell viability of cultured DPSCs on these scaffolds significantly increased. Also, the anti-inflammatory and anti-oxidative capabilities of chrysin-based scaffolds were evidenced by the decreased levels of TNF


Bispectral index (BIS) and response entropy (RE) are used to monitor the depth of anesthesia.To collect published data and compare the accuracy of BIS and RE in detecting the transition of consciousness during sevoflurane anesthesia.Studies indexed in the PubMed, Embase, or Cochrane databases.1.Monitoring of sevoflurane anesthesia depth with BIS and RE simultaneously;2.Use of prediction probability values to evaluate prediction accuracy; and3.The full text of the published study is available and contains sufficient data for further analyses.Patients who need to use BIS and RE to monitor sevoflurane anesthesia depth simultaneously.I2 methodology was used to determine the heterogeneity of the statistical results, while GRADE Pro served to assess the quality of evidence.A random-effects model was fitted using RevMan 5.3. Subgroup analyses were performed on patient age. The Cochrane P = .009; I2 = 92%). In contrast, there was no significant difference between BIS and RE for recovery of consciousness . Subgroup analyses revealed no significant differences in LOC  and ROC  in children. However, the results in adults demonstrated that BIS is more accurate than RE in predicting LOC .Overall, 195 articles were identified, of which 7 were finally included. The meta-analysis results showed that BIS is more accurate than RE in predicting loss of consciousness (LOC) during sevoflurane anesthesia (MD, .06; 95% confidence interval [CI], .02–.09; First, this meta-analysis was affected by a large study heterogeneity. Second, this analysis only included publications in English, therefore, some studies may have been omitted.BIS is more accurate than RE in predicting LOC during sevoflurane anesthesia in adults. However, no significant differences were identified in children.CRD42020163119 RE values also range between 0 and 100 .Changes in consciousness during anesthesia are a very important aspect related to the mechanisms of anesthesia. The bispectral index (BIS) and response entropy (RE) are both electroencephalography (EEG)-derived methods to monitor the depth of anesthesia. However, the examination method, can strongly influence the results. Although several studies tried to determine the accuracy of BIS and RE in distinguishing the transitions of consciousness, this remains an open question. Addressing this problem may benefit clinical research.The transitions of consciousness during anesthesia comprises 2 parts: anesthetic-induced loss of consciousness (LOC) and recovery of consciousness (ROC). Traditionally, both have been assessed by observing responses to verbal commands.The prediction probability value (PK) has been used in many studies to assess how accurately BIS and RE distinguish consciousness transitions. A PK of .5 represents a 50% success rate to predict the observed state, whereas a PK of 1.0 indicates that this method always predicts the correct state.1.2This meta-analysis aimed to collect published data and compare the accuracies of BIS and RE to detect changes in consciousness during sevoflurane anesthesia.22.1This study conforms to the 2009 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, and was registered in PROSPERO (CRD42020163119). Because all analyses were based on previously published studies, no ethical approval or patient consent was required. All data were analyzed anonymously during the review process. The methods of analysis and inclusion criteria were specified in advance and documented in the protocol. The inclusion criteria for this meta-analysis were as follows:1.Simultaneous monitoring of sevoflurane anesthesia depth with BIS and RE;2.PK use to evaluate prediction accuracy;3.The full text of the published study was available, and the data sufficient for further analyses.The exclusion criteria for this meta-analysis were:1.animal studies and2.lack of adequate data.No constraints on language, publication date, or publication status were imposed.2.2Two authors (TL and FW) independently searched the following databases for relevant literature: PubMed, Embase, and the Cochrane Library. Systematic reviews published until October 2019 were included. The following keywords were used in the title and abstract fields: “sevoflurane,” “anesthesia, general,” “bis,” “bispectral index,” “RE,” and “entropy.” The electronic search strategy for the 3 databases was defined as follows: ((((“sevoflurane” [MeSH Terms] OR “sevoflurane” [All Fields]) OR “sevoflurane” [All Fields]) OR “sevoflurane s” [All Fields]) AND ) AND ((((“radiation effects” [MeSH Subheading] OR (“radiation” [All Fields] AND “effects” [All Fields])) OR “radiation effects” [All Fields]) OR “re”[All Fields]) OR ((“entropy” [MeSH Terms] OR “entropy” [All Fields]) OR “entropies” [All Fields])).2.3The eligibility assessment of the retrieved studies was independently performed in an unblinded standardized manner by 2 reviewers (TL and FW). Disagreements between reviewers were resolved by consensus. The same 2 investigators independently extracted data from publications. A third author (FM) resolved any discrepancies arising during the data extraction and evaluation process. The following information was extracted from each included trial:1.Trial characteristics including age, sample size, year of publication, and authors;2.Prediction probability values for RE and BIS;3.Information regarding the monitor brand; and4.Methods of consciousness evaluation and reasons for removing data from the present analysis.2.4The risk of bias in the included studies was assessed using the risk of bias table in RevMan 5.3. Two authors (TL and FW) answered 7 questions regarding selective reporting, incomplete outcome data, random sequence generation, allocation concealment, blinding of participants, and outcome data to evaluate the risk of bias. The impact of bias on a study was divided into 3 levels: low, high, and unknown risk. For each trial, we plotted the effects against standard errors. The symmetry of the resulting “funnel plot” was visually examined to assess the presence of publication bias in the outcome indicators.2.5Two reviewers (TL and FW) independently evaluated the quality of evidence in accordance with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology. Assessment items included study design, risk of bias, inconsistency, indirectness, imprecision, and other considerations. The results are divided into different levels . GRADE Pro was used to construct summary tables for the included studies.2.6I2 methodology was used to evaluate the heterogeneity of the statistical results . Statistical significance was set at P < .05. Subgroup analysis was performed on the patients’ age.The primary outcome measure of this analysis was the PK of both BIS and RE to detect changes in consciousness during sevoflurane anesthesia. Data in the current study are presented as mean ± standard deviation. The meta-analysis was performed using RevMan 5.3, with fitting of a random effect model. The Cochrane 33.1The initial database search rendered 195 articles. After identifying duplicate records, 121 studies were excluded. After reading titles and abstracts, 63 studies were further excluded. After reading the remaining full-text articles in more detail, 7 studies were finally included in this meta-analysis . A flowchart of the publication selection process is shown in Figure 3.2All 7 studies selected were published in English. A total of 248 samples were included in the selected studies. Five studies collected data from adult patients, whereas 2  analyzed children data. All studies used the following monitors: BIS  and RE , except for 1 study which used a different device to monitor BIS . One study used the observer's assessment of alertness/sedation scale (OAA/s) to evaluate LOC and ROC , 1 study did not mention the methodology , and the remaining 5 studies evaluated LOC and ROC by observing patients’ reactions to verbal commands. More information is shown in the overview table, including induction drugs, opioid use, neuromuscular blocker use, type of surgery, and publication date (Table 3.3I2 = 92%), further evidence by the symmetry of the corresponding funnel plot , but there was no significant difference between BIS and RE in children  or children . The results of this subgroup analysis suggested no significant differences between BIS and RE either in adults  which may increase the differences of results. Two studies included in this analysis included data from children. Although the BIS has been validated in children, its validity in infants is less clear. Some research supports RE's potential to be less influenced by age since it is independent of amplitude and frequency. Klockars et al reported better PKs in children than in infants and also better during induction than emergence. Further, PKs in both age groups were lower than in adults, and most evident in infants. Interestingly, this is also the case for BIS. The study by Davidson et al suggests that BIS and entropy similarly reflect the effect of sevoflurane anesthesia in children, although age has a profound impact on the accuracy of both methods, with better accuracies in increasing age. The results of Ellerkmann et al also confirmed previous evidence that, in infants, BIS and entropy monitors are less linearly correlated. In our meta-analysis, low performance parameters indicate that both BIS and RE fail to capture the state of anesthesia in infants.EEG activity changes with age, therefore this is an important aspect to consider.4.2This meta-analysis had some limitations. First, it was affected by a large study heterogeneity. This may be caused by small sample sizes, differences in protocol design and monitor devices, anesthetic procedures, and surgery types. Second, this analysis only included publications in English; therefore, some studies may have been omitted.4.3The change in consciousness during anesthesia is an important aspect when studying the mechanisms of anesthesia. The results of this meta-analysis suggest that BIS is more accurate than RE for detecting LOC during sevoflurane anesthesia in adults. Given the heterogeneity of the studies included, caution must be prevailed, especially when extrapolating to infant populations.Data curation: Feng Mu.Formal analysis: Tao Liang, Fan Wu.Funding acquisition: Feng Mu.Methodology: Feng Mu.Project administration: Fan Wu, Baoguo Wang.Resources: Fan Wu.Software: Tao Liang.Supervision: Baoguo Wang.Writing – original draft: Tao Liang.Writing – review & editing: Baoguo Wang.


To evaluate the effect of nicotinamide prior to streptozotocin-induced (STZ) diabetes in baroreflex sensitivity and cardiovascular autonomic modulation, and its association with hemodynamics and metabolic parameters.Methods: Male Wistar rats were divided into control (Cont) and STZ-induced diabetes (Diab). Half of the rats from each group received a single dose of nicotinamide (100 mg/Kg) before STZ injection (Cont+NicA and Diab+NicA). All groups were followed-up for 5 weeks.Body weight loss of more than 40% was observed in Diab throughout the period . Increased glycemia was seen in Diab rats (541.28 ± 18.68 mg/dl) while Diab+NicA group had a slight decrease (440.87 ± 20.96 mg/dl). However, insulin resistance was observed only in Diab. In relation to Cont, heart rate, mean blood pressure and diastolic function were reduced when compared to Diab, together with parasympathetic modulation and baroreflex sensitivity. All of these parameters were improved in Diab+NicA when compared to Diab. Improved baroreflex sensitivity and parasympathetic modulation were correlated with glycemia, insulin resistance, and body weight mass. Additionally, Diab+NicA group increased survival rate.Results suggest that the association of nicotinamide in STZ-induced diabetic rats prevents most of the expected derangements mainly by preserving parasympathetic and baroreflex parameters. Diabetes mellitus (DM) is a complex metabolic disease affecting about 422 million people worldwide. It is characterized by chronic hyperglycemia, insulin resistance, and/or insulin secondary deficiency caused by the failure of β-pancreatic cells , 2.According to the classification proposed by the American Diabetes Association (ADA), there are two DM types: type 1 diabetes and type 2 diabetes are heterogeneous diseases in which clinical presentation and disease progression may vary considerably . Type 2 Different experimental models emerged as possibilities to mimic human types 1 and 2 DM to study its development and complications. Masiello et al. 1998) developed a rat model of diabetes induced by administration of streptozotocin (STZ) and nicotinamide. STZ selectively destroys pancreatic beta cells, while N decrease the damage caused by STZ, creating a state of partial insulin deficiency, similar to what occurs in type 2 diabetes. The severity of STZ + N-induced diabetes is much lower than that of diabetes induced by STZ alone; rats manifest moderate hyperglycemia and do not require exogenous insulin to survive [ developeNicotinamide, a derivative of vitamin B3, effectively protects β-cells against the cytotoxicity of STZ. The protective action of N relies on the decreased damage and deoxyribonucleic acid (DNA) methylation in pancreatic islets, improved insulin secretion , 11, neuAlthough all of these mechanisms play a pivotal role in diabetes development, autonomic nervous system also underlies cardiometabolic pathophysiology in metabolic diseases. Studies in fructose-fed animals demonstrated that autonomic nervous system dysfunction, mainly represented by baroreflex sensitivity impairment, not only accompanies the disease progression but may also precede metabolic and hemodynamics alterations , 14. LinSince autonomic control of circulation seems to modulate DM complications, and nicotinamide presents a protective role in diabetic rats induced by STZ, we hypothesized that preserved cardiovascular autonomic modulation and baroreflex sensitivity could also mediate the attenuation of STZ-induced derangements in rats pre-treated with nicotinamide. Therefore, this study aimed to evaluate the effect of nicotinamide prior to STZ-induced diabetes in baroreflex sensitivity and cardiovascular autonomic modulation, and its association with hemodynamics and metabolic parameters.This research was carried out in accordance with the ARRIVE procedures for animal experiments . All expOn the day of diabetes induction, all rats underwent a 6-h fasting. Cont+NicA and Diab+NicA rats received a single intraperitoneal injection of 100 mg/kg of nicotinamide (Sigma-Aldrich) 15 min before diabetes induction , 19. DiaAll animals were weighed once per week throughout the protocol period. To determine the body mass index, Lee index was calculated by cube root of body weight (g) × 10/naso-anal length (mm) , 22.At the beginning and at the end of the protocol, all animals underwent 4 h of fasting. A drop of blood from the tail vein was collected to measure plasma glucose using a glucometer, and another drop was collected for triglyceride measurement with Accu-Chek e Accutrend GTC , respectively.ITT was performed at the end of the protocol. Baseline glucose levels were determined according to Furuya et al. 2003. Insulin (0.75 units/kg) was injected subcutaneously. Then, blood samples were collected from the tail vein at 0 (just before the insulin injection), 4, 8, 12 and 16 min (after the insulin injection) for the glucose assay. Plasma glucose was measured from blood samples collected from the tail vein with the use of a glucometer  –25. GlycEchocardiographic evaluations were blindly performed by an expert and in accordance to the guidelines of the American Society of Echocardiography. Rats were anaesthetized  and images were obtained with a 10–14 mHz linear transducer in a SEQUOIA 512  for measurements of left ventricular function and morphology. Echocardiographic parameters were measured as previously described in detail elsewhere , 28.One day after the echocardiographic evaluation, two polyethylene catheters filled with 0.06 ml of saline were implanted into the femoral artery and femoral vein (PE-10). Rats were anesthetized with isoflurane (1 a 2.5%), for direct measurements of arterial pressure and drug administration, respectively. After this procedure, the animals were allowed to recover in individual cages. The rats were taken to the recording room at least 30 min before the beginning of the experiment, and a quiet environment was maintained to avoid any stress. On the next day, the arterial cannula was connected to a strain-gauge transducer , and arterial pressure signals and pulse interval (heart rate) were digitally recorded over a 30-min period in conscious animals, moving freely during the experiments using a data acquisition system  as previously described , 29. BeaFor baroreflex sensitivity evaluation, sequential bolus injections of increasing doses of phenylephrine (0.25 to 32 μg/kg) and sodium nitroprusside (0.05 to 1.6 μg/kg) were given to increases or decreases in mean arterial pressure responses (for each drug), ranging from 5 to 40 mmHg.32. Peak increases or decreases in mean arterial pressure after phenylephrine or sodium nitroprusside injection and the corresponding peak reflex changes in heart rate were recorded for each dose of the drug. A 3–5 min interval between doses was necessary for arterial pressure to return to baseline. Baroreflex sensitivity was expressed as bradycardic response and tachycardic response, expressed as bpm/mmHg, as described elsewhere in beats per minute per millimeter of mercury , 29, 30.The heart rate variability was performed by linear methods in time and frequency domain by Cardioseries® v2.4. Temporal series of pulse interval and systolic blood pressure from baseline recordings were analyzed and total heart rate variability (HRV) and systolic arterial pressure variability (SAPV) were calculated. The mean square root of differences between consecutives PI (RMSSD), an index of parasympathetic modulation in time domain, was also evaluated. For frequency domain, the interpolated wave of these same basal periods were divided in segments of 512 beats with overlap of 50% and processed by Fast Fourier Transform. One spectrum was obtained by each segment and the oscillatory components were quantified in low frequency , which indicates sympathetic modulation predominance, and high frequency , which indicates parasympathetic modulation predominance .p < 0.05.All data are expressed as mean ± SEM. The two-way analysis of variance followed by Newman-Keuls post hoc test was used to compare groups. Repeated measures two-way analysis of variance was used when necessary. Pearson’s correlation was used for the study of associations between variables. Kaplan-Meier method was used to calculate survival rate and curves were compared by logrank test. Statistical calculations were performed using Graph Pad Prism version 6.0. Significance level was established at p < 0.05) . Similarly, gastrocnemius muscle weight was also lower in diabetic groups  when compared to controls . Absolute cardiac mass was not different among groups .At the end of the protocol Cont and Cont+NicA groups presented body weight gain in relation to initial values. In contrast, Diab group presented body weight loss throughout the protocol, and nicotinamide prevented body weight loss in diabetic rats Fig. A. MoreovBefore induction, all groups had similar glycemia values (week 0) Fig. A. As expResults from hemodynamic and cardiovascular autonomic modulation are presented in Table Results of baroreflex sensitivity evaluated by heart rate changes to vasoactive drugs revealed a reflex impairment of the bradycardic response in Diab vs. Cont. nicotinamide treatment in STZ rats increased both tachycardic and bradycardic responses in relation to Diab Fig. .Fig. 3BEchocardiography analysis results are shown in Table Mortality among studied rats was evaluated for 5 weeks. Our results showed that nicotinamide was an effective intervention to increase survival rate in STZ-induced diabetic rats. Figure p = 0.003), and the rate constant for plasma glucose disappearance (kITT) . In addition, RMSSD was also associated with kITT . Moreover, body weight gain was associated with both bradycardia and tachycardia indexes with , as well as with kITT  and RMSSD .Pearson’s correlation analysis using all diabetic rats showed strong associations between the index of reflex bradycardia and fasting glycemia . Guo et al. (2019) reported that nicotinamide had a protective effect on skeletal muscle atrophy in STZ-induced diabetic mice, which may be through inhibition of TGF-b1/Smad2 pathway . PreservAltogether, the improvements induced by nicotinamide in diabetic rats resulted in increased survival rate. In 13 weeks, survival in STZ-induced diabetic rats was approximately 50% . In our Diabetes induced by STZ and nicotinamide remains stable for a long time and thus this model of diabetes is suitable not only for short-term but also for long-term studies. Moreover, this model is very useful in investigations of different aspects of diabetes, including diabetic complications and anti-diabetic properties of new drugs and natural compounds .Nicotinamide associated with STZ is a more stable model of experimental diabetes by preventing STZ-induced baroreflex impairment, parasympathetic dysfunction and insulin resistance in rats, independently of glycemia levels. In association with improved metabolic profile, and preserved body weight mass, these protective effects of nicotinamide resulted in increased survival rate.


Bisphenol A (BPA) is an endocrine disruptor that it is present in numerous products of daily use. The aim of this study was to assess the potential association of serum BPA concentrations and the risk of incident breast and prostate cancer in a sub-cohort of the Spanish European Prospective Investigation into Cancer and Nutrition (EPIC).We designed a case-cohort study within the EPIC-Spain cohort. Study population consisted on 4812 participants from 4 EPIC-Spain centers . BPA exposure was assessed by means of chemical analyses of serum samples collected at recruitment. Borgan II weighted Cox regression was used to estimate hazard ratios.p = 0.022), 37% increase for tertile 2 (p = 0.034) and 31% increase for tertile 3 (p = 0.072) was observed with respect to values bellow the limit of detection. No significant association was observed between BPA levels and breast cancer risk.Median follow-up time in our study was 16.9 years. BPA geometric mean serum values of cases and sub-cohort were 1.12 ng/ml vs 1.10 ng/ml respectively for breast cancer and 1.33 ng/ml vs 1.29 ng/ml respectively for prostate cancer. When categorizing BPA into tertiles, a 40% increase in risk of prostate cancer for tertile 1 (We found a similar percentage of detection of BPA among cases and sub-cohort from our population, and no association with breast cancer risk was observed. However, we found a higher risk of prostate cancer for the increase in serum BPA levels. Further investigation is needed to understand the influence of BPA in prostate cancer risk.The online version contains supplementary material available at 10.1186/s12940-021-00779-y. Cancer incidence is increasing worldwide with 18.1 million new cancer cases and 9.6 million cancer deaths in 2018 . Some riBPA is considered a non-persistent chemical, i.e., as it is degraded in the organism and there is evidence that BPA acts as an endocrine disruptor with estrogenic effects in the rodent mammary gland –27. MoreThe present study aims to assess the potential association of serum BPA concentrations and the risk of incident breast and prostate cancer in a sub-cohort of the Spanish European Prospective Investigation into Cancer and Nutrition (EPIC).We designed a case-cohort study within the EPIC-Spain cohort. EPIC is a prospective multi-centric cohort study planned to investigate the relationship between diet, lifestyles and cancer. It involves 23 research centers in 10 European countries, including five Spanish centers: Asturias, Granada, Murcia, Navarra and Gipuzkoa . Study pThe EPIC-Spain included 41,446 participants (62% women) aged 29–69 years enrolled between 1992 and 1996 in five provinces of Spain. Participants were recruited mostly among blood donors (about 60%) and the study population covered a broad range of socioeconomic and educational levels. Furthermore, they signed an informed consent and the study was approved by Ethics Committee of the Bellvitge Hospital (Barcelona). EPIC study populations and data collection were explained elsewhere [Study population in the present study consisted of 3690 sub-cohort participants, 547 breast cancer cases and 575 prostate cancer cases from four EPIC-Spain centers  with available data on BPA exposure. Participants selected for the sub-cohort included, by design, an overlap of 57 breast cancer cases and 111 prostate cancer cases  and age (quintiles), excluding persons with cancer at recruitment. In our sub-cohort, 79.6% of the participants provided a fasting blood sample at recruitment and 85.6% of them were extracted between 6 am and 11 am. However, we can generalize and assume most of our samples were taken during the mornings and in fasting conditions.Follow-up time began at EPIC recruitment and cancer cases were defined as participants with a diagnosis of breast or prostate cancer (i.e. ICD10 codes C50 and C61 respectively) during the study period. Incident cancer cases are identified by linkage with the Population Cancer Registries. Incidence date was determined by the date of cancer diagnosis and prevalent cases were excluded (participants with a cancer diagnosis prior to recruitment). Dates of end of follow-up for cases identification were: 31/12/2012 for Granada, 31/12/2013 for Murcia, 31/12/2011 for Navarra and 30/12/2013 for Gipuzkoa. For sub-cohort participants, the end of follow-up date was the lowest among date of diagnosis, date of death, date of loss of follow-up or the center-specific end-of-follow-up, whatever happened first.Vital status and date of death of participants in our sub-cohort was determined by linking EPIC data base with the National and regional Registries, IND , INE  and regional mortality register until 30/12/2013 (maximum date of identification of incident cases).Information on lifestyle and other health-related factors was obtained by an interviewer-administered questionnaire at baseline. All interviewers had received appropriate training for this task.2, 25- < 30 kg/m2, ≥30 kg/m2. Educational level was classified according to five categories: none, primary school, secondary school, technical or vocational training and university degree. Smoking status was summarised in three categories: never smoked, former smoker and current smoker. Alcohol consumption at recruitment in grams per day was categorized as no drinker (0 g/day), drinker (≤30 g/d in men and ≤ 20 g/d in women) and heavy drinker (> 30 g/d in men and > 20 g/d in women). Information on the domains of physical activity was compiled taking seasonal variation into account. A simple four-level physical activity index  was derived and validated by combining recreational and household activity [Measurements of height, weight, and hip and waist circumferences were taken at recruitment using standardised procedures . The queactivity . ReproduBlood samples were drawn from each participant at recruitment, which were subsequently centrifuged, and aliquots of plasma, serum, red blood cells and buffy coat in 0.5 mL straws were stored in liquid nitrogen (− 196 °C).BPA levels were quantified in serum samples using two of 0.5 mL straws, in an adaptation of a previously-validated methodology . In briehttps://www.medinadiscovery.com/, which has been assessed and certified for the standards of ISO 9001:2015, and routinely performs internal and external quality control analyses. In addition, the present analyses are encompassed in the activities of our research group within the The Human Biomonitoring Initiative .Chemical analyses were performed at Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía MEDINA  were calculated for cases and sub-cohort and according to center, sex, age group, educational level, body mass index, physical activity and alcohol consumption and smoker status. Statistical differences were assessed through the Mann-Whitney or Kruskall-Wallis tests.Time to cancer event were modelled by means of Borgan II weighted Cox proportional hazard models , stratifIn our study, median follow-up time was 17 years. We had 547 cases of breast cancer and 575 prostate cancer cases and 3690 sub-cohort participants (1918 women and 1772 men). Table Among breast cancer patients, there were more smokers (18.1% vs 10.3%) than in the sub-cohort. Regarding physical activity, the sub-cohort had more participants in the “very high” classification than the cases: 58.7% vs 50.3% respectively. Other significant differences were observed in education level, BMI, menopausal status, number of pregnancies, breastfeeding and oral contractive use comparing breast cancer cases and sub-cohort participants (Table p = 0.754)  between smoker . However, there were no significant differences when observing other variables, neither those concerning women reproducibility , neither in logarithmic model  . Moreover, when categorizing BPA in tertiles we observed an increased risk of prostate cancer in model C: a 40% increase in risk of prostate cancer for tertile 1 , 37% increase for tertile 2  and 31% increase for tertile 3  was observed with respect to values bellow the limit of detection (p-trend = 0.069)  were no significant differences in blood BPA levels between breast cases and controls were found (p = 0.42) [In our study, 70% of the population had detectable BPA values and its concentrations were similar among cancer cases and sub-cohort participants which is comparable to an epidemiological study in Korea ( = 0.42) .n = 1000 participants) [n = 575) [n = 264) [Our results show no association for BPA serum concentrations and risk of incident breast cancer despite BPA is considered an ED with carcinogenic potential –33. Thescipants) . In the n = 575) . Howevern = 575) , 47–49. n = 264) . Mammogrn = 60) showed a much higher concentration of BPA in the urine of those patients in comparison with the control group [Regarding prostate cancer, we found a significant non-linear association with risk of prostate cancer when we categorized serum BPA in tertiles, as individuals with serum BPA levels in the 1st and 2nd tertile showed a 40 and 37% increased risk of prostate cancer respectively. In this regard, a case control study conducted in men with prostate cancer  (percentage above the limit of detection (LOD) and geometric mean (GM) with 95% confidence interval) by sociodemographic and life style characteristics in breast cancer cases and sub-cohort. Table S2. Serum BPA levels (ng/ml) (percentage above the limit of detection (LOD) and geometric mean (GM) with 95% confidence interval) by sociodemographic and life style characteristics in prostate cancer cases and sub-cohort.


Salvia miltiorrhiza, has many activities, such as anti-inflammatory and antisepsis activities. However, the role of PCA in osteoclasts is not clear. We used RAW264.7 cells (a mouse leukemic monocyte/macrophage cell line) and bone marrow macrophages (BMMs) to probe the role of PCA in osteoclasts and the underlying mechanism. The effects of PCA on cell activity were evaluated with CCK-8 assays. TRAP staining detected mature osteoclasts. Corning Osteo Assay Surface plates were used to examine absorption. The levels of RNA and protein were analyzed, respectively, using RT-PCR and Western blotting. PCA (5 μg/ml) was not toxic to the two cell types but reduced the formation of osteoclasts and bone absorption. Furthermore, PCA restrained the expression of mRNAs encoding proteins associated with osteoclasts and reduced the phosphorylation of proteins in important signaling pathways. The results indicate that PCA inhibits osteoclast differentiation by suppressing NF-κB and MAPK activity.Protocatechualdehyde (PCA), an important component of  Osteoporosis is a degenerative disease with an increasing risk due to aging and is one of the most common diseases in older men . The disUnder normal circumstances, bone tissue maintains normal function by constantly updating metabolism. Due to abnormal metabolism, older patients experience bone loss. The increased activity of osteoclasts increases the loss of bone mass, and the attenuated function of osteoblasts decreases the formation of bone mass, leading to fractures in the bone microstructure and a negative balance in bone reconstruction . TherefoCurrently, bisphosphonates are common agents used to treat osteoporosis and significantly inhibit the activity of osteoclasts. They have been recommended by many authoritative institutions worldwide due to their clear efficacy . HoweverSalvia miltiorrhiza (SM) is a Chinese herbal medicine that has long been used to treat osteoporosis. Clinical trials have shown that SM alone is as effective as traditional drugs in the treatment of osteoporosis and has the advantage of being nontoxic [In vitro experiments have confirmed that SM exerts a good protective effect on ovariectomy-induced osteoporosis and diabetic osteoporosis [nontoxic . In vitroporosis , 7. Salvoporosis , increasIn vivo, the prodrug PCA restrains the destruction of the femoral condylar cartilage caused by adjuvant arthritis [Protocatechualdehyde (PCA) is the main degradation product of salvianolic acid B and has many activities, such as anti-inflammatory and antibacterial properties . In vivorthritis . PCA sigrthritis . HoweverIn this study, RAW264.7 cells (a mouse leukemic monocyte/macrophage cell line) and bone marrow macrophages (BMMs) were used to analyze the role of PCA in osteoclasts and the underlying mechanism. PCA has important theoretical significance and clinical application value in the search for economical and effective drug monomers to treat osteoporosis.RAW264.7 cells were selected from ATCC . BALB/c mice were purchased from Shandong University. PCA (purity > 98% by HPLC) was obtained from Sigma. Cell culture media were obtained from BI. Cytostimulators were acquired from R&D Systems. CCK-8 and TRAP kits were purchased from Sigma. 24-well bone plates were purchased from Corning Life Science . Phalloidin was obtained from Thermo Fisher Scientific. The apoptosis kit was purchased from BD. All antibodies were acquired from Cell Signaling Technology . Primers were designed by PrimerBank and synthesized by Shanghai Bioengineering Company.α-MEM, and red blood cells were removed to obtain BMMs. The lysate was filtered through a 40 μm mesh filter and centrifuged to separate the cells. Samples from the mice were cultured overnight in α-MEM containing M-CSF (5 ng/ml) in a nonadherent culture plate, and then, cells suspended in the collected supernatant were cultured with M-CSF (30 ng/ml) for 3 days. The supernatant and suspended cells were discarded; the adherent cells were the BMMs.BMMs were acquired from both ends of cut femurs and tibias from approximately 6-week-old male BALB/c mice. The animal experiment was authorized by the Experimental Animal Ethics Review Committee of Shandong University. The marrow cavity was rinsed 3-5 times with α-MEM to culture osteoclasts induced by receptor activator of nuclear factor-κB ligand (RANKL).RAW264.7 cells were cultured in complete DMEM. However, DMEM was substituted for 4 cells/well) and RAW264.7 cells (1 × 104 cells/well) were seeded in the presence of PCA  for 5–6 days. CCK-8 (10 μl/well) was added to the supernatant and incubated for 0.5–1 h, and the absorbance was assayed at 452 nm by a Multiskan Go1510 microplate reader. The ratio of the experimental group to the control group represents cell activity [CCK-8 assays were used to determine the toxicity of PCA toward cells. In 96-well plates, BMMs (2 × 10activity .3 cells/well) were cultured in 48-well plates, and BMMs (2 × 104 cells/well) were seeded in 96-well plates. The next day, 50 ng/ml RANKL and 30 ng/ml M-CSF were added to the BMM culture media, and 50 ng/ml RANKL was added to the RAW264.7 culture media. In addition, PCA  were added to the medium. After 4–6 days, the cells were stained and identified according to the literature [RAW264.7 cells (5 × 10terature .5 cells/well) and RAW264.7 cells (1 × 105 cells/well) were seeded and incubated for 24 h. Next, 50 ng/ml RANKL and 30 ng/ml M-CSF were added to the BMM culture media, and 50 ng/ml RANKL was added to the RAW264.7 culture media. In addition, PCA  was added at the same time, and the cells were incubated for 4–8 days. After 4–8 days, a 10% bleach solution was used to wash the cells. A light microscope (Olympus) captured images of the absorption pits, and ImageJ software quantified the image [We used Corning Osteo Assay Surface 24-well plates to assay absorption pits. BMMs (4 × 10he image .5 cells/well) were seeded in 24-well plates, and a series of PCA, M-CSF (30 ng/ml), and RANKL (50 ng/ml) were added to the medium. The cells were incubated for 4 days, and phalloidin was used to stain the cells after fixation and permeabilization. A fluorescence microscope was used to capture fluorescence images.BMMs (3 × 105 cells/well) were seeded in 6-well plates and cultured with a series of PCA and 50 ng/ml RANKL. After 3–4 days, the apoptosis kit was used to stain the cells. Cell apoptosis was assessed using flow cytometry.RAW264.7 cells (5 × 104 cells/well). The next day, the cells were pretreated with a series of PCA concentrations for 60 min before adding 50 ng/ml RANKL. After 5–6 days, the cells were collected. PCR was performed as previously described [RAW264.7 cells were seeded in 24-well plates (5 × 10escribed . Primers6 cells/m) were plated in 6-well plates and then pretreated with PCA  for 2 h before the addition of RANKL (50 ng/ml) and incubation for 10 min. Western blots were performed as previously described [RAW264.7 cells  has no effect on cell activity.The potential cytotoxicity of PCA toward RAW264.7 cells  and BMMsBMMs and RAW264.7 cells were successfully induced to differentiate into osteoclasts by RANKL. The osteoclasts were large and full, with vacuoles forming in the middle and additional nuclear accumulation throughout the cells. However, when PCA was present, the size of the osteoclasts decreased, the vacuoles disappeared, and the number of surrounding nuclei decreased. Furthermore, we used TRAP to stain osteoclasts. The test results indicated that PCA prevented osteoclastic differentiation not only in BMMs but also in RAW264.7 cells Figures . Thus, Pμg/ml PCA to the medium and induced BMMs to differentiate in three periods to determine the stage of osteoclastogenesis at which PCA exerted its effect. Osteoclast formation was markedly inhibited by PCA in the earlier period  to study the roles of PCA in the induction and absorption function of osteoclasts . Throughin vitro; thus, CTSK is an important factor for determining osteoclast function [κB in the incipient stage of osteoclast formation. In vitro experiments confirmed that stem cells lacking NFATc1 fail to differentiate into osteoclasts, but ectopic NFATc1 expression in the absence of RANKL also enables BMMs to differentiate into osteoclasts [κB signaling pathways.A series of iconic factors are produced in the course of differentiation from precursor cells to mature osteoclasts and thus can be used as markers of osteoclasts and their differentiation stages. Cathepsin K (CTSK) is an amino acid protease expressed by osteoclasts. CTSK stimulates the expression of TRAP on osteoclasts function . The receoclasts . These reoclasts . Dendriteoclasts . Cellulaeoclasts . In the MAPK is an important signaling pathway involved in osteoclast differentiation . This paκB signaling is another indispensable pathway in osteoclast induction [κB. Then, NF-κB separates from I-κB and is quickly transported into the nucleus, binding to the promoters of the corresponding target genes and regulating osteoclast differentiation, maturation, and apoptosis [κB activity. PCA possibly inhibits the transcription of NFATc1 by restraining the phosphorylation of NF-κB p65, thereby affecting the expression of downstream genes.NF-nduction . The tripoptosis , 31. In PCA is a water-soluble component of SM. SM and its other active ingredients are known to possess antiosteoporosis properties. Tanshinone is one of the most much-studied active components at present. Published studies have shown that tanshinone inhibits osteoclast activation and reduces bone absorption by destroying F-actin rings . CryptotκB? Does PCA bind to cognate receptors on the cell surface of precursor cells and subsequently inhibit cellular signaling pathways that lead to NF-κB and MAPK activation, or does PCA freely enter cells and directly inhibit NF-κB and MAPK activation? Other questions include the effectiveness of PCA in vivo and its pharmacokinetics, which all require further study.Although some results were reported in our study, many other issues remain to be further explored. For example, how does PCA suppress the activation of MAPK and NF-κB activation. Therefore, PCA potentially represents a cost-effective and alternative medicine for osteoporosis.In conclusion, our research shows that PCA suppresses osteoclast differentiation by inhibiting MAPK and NF-


Decreased physical activity had been reported to be a common causal and modifiable risk factor for major vascular events. However, the relationship of physical activity and sedentary leisure time with carotid atherosclerosis in population with high risk for cardiovascular diseases (CVDs) is still inconclusive. We aimed to evaluate the association of physical activity and sedentary leisure time with the risk of carotid atherosclerosis, and investigate any possible effect modifiers in population with high risk for CVDs.The study population was drawn from the China Patient-Centered Evaluative Assessment of Cardiac Events (PEACE) Million Persons Project-Jiangsu project, which is a population-based screening project that included permanent residents aged 35-75 years from 6 surveillance cities in Jiangsu Province. Linear regression models were used to evaluate the association of physical activity and sedentary leisure time with carotid intima-media thickness (CIMT). The risks of abnormal carotid artery and carotid plaque (CP) were estimated by odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression.P<0.05 for all). Furthermore, the inverse association were stronger in participants with age ≥60 years . However, there were no significant association of sedentary leisure time with CIMT, abnormal carotid artery and CP.Overall, a total of 10,920 participants were enrolled in the final analysis. There was a significant inverse association of physical activity level with CIMT . The risk of abnormal carotid artery and CP decreased significantly with the increase of physical activity level . When physical activity level was categorized as quartiles, a significantly lower risk of abnormal carotid artery and CP was found in quartiles 2-4 with quartile 1 as reference (In population with high risk for CVDs, physical activity was inversely associated with CIMT, abnormal carotid artery and CP, particularly among the elders. Sedentary leisure time was not associated with them. These results suggested that physical activity is important for carotid vascular health, and perhaps especially in elder population.The online version contains supplementary material available at 10.1186/s12889-022-12582-6. In recent years, cardiovascular diseases (CVDs) is the leading cause of disability and premature death worldwide, representing a severe public health problem . By 2030Increasing physical activity level and reducing sedentary leisure time had been reported to be common causal and modifiable risk factors for major vascular events , 8. ThisTherefore, we aimed to explore whether there is an association of physical activity level and sedentary leisure time with CAS assessed by CIMT, CP and abnormal carotid artery, and to examine the possible effect modifiers in population with high risk for cardiovascular diseases (CVDs) in China.A government-funded public health program was conducted in 2015 in Jiangsu province, which is part of China PEACE  Million Persons Project , launcheDuring the initial screening stage, each participant received face-to-face questionnaire interview, physical examination and laboratory measurement.2). Seated blood pressure was measured twice on the right upper arm after 5 min of rest with an automatic digital sphygmomanometer . If the difference between the two SBP readings was greater than 10 mmHg, a third measurement was obtained and the average of the last 2 readings was used. Fasting vein blood glucose test was performed by a rapid glucose analyzer  and total cholesterol (TC), triglyceride (TG), HDL-C and LDL-C is performed by a rapid lipid analyzer . LDL-C is calculated using the values of TC, HDL-C and TG levels in the Friedewald equation.The questionnaire included information on: (1) sex, age and other general demographic data; (2) smoking and drinking status; (3) education and annual household income; (4) history of diseases and treatment. Anthropometric measurements included height, weight and waist circumference (WC). Participants were required to wear light clothes, with no shoes and no headgear while being measured for height and weight. Body mass index (BMI) was calculated by dividing weight (kg) by height  , and CKBParticipants were asked how many hours per day they spent on sedentary activities during leisure-time such as watching television, reading, playing card games and weaving was defined as sedentary leisure time.All carotid ultrasound examinations were obtained by registered ultrasound physicians  who have worked for at least 5 years. Ultrasound assessment technique followed the recommended guidelines for ultrasound measurement approved by National Center for Cardiovascular Disease. CIMT (mm) were measured for both the right and left common carotid arteries using ultrasound scanner equipped with a variable frequency (5-12 MHz) linear array transducer. Bilateral carotid arteries were scanned with the common carotid artery bifurcation and the near and far walls of the distal 1-1.5 cm proximal to its bifurcation. The IMT of posterior wall with the minimum diameter of carotid artery in diastolic period was used. CIMT values were calculated as the average of three areas on both the right and left carotid artery. Abnormal carotid artery was defined as CIMT ≥1.0 mm at any measurement position , and CP Characteristics were presented as means (standard deviation) for continuous variables and proportions for categorical variables. The differences characteristics between abnormal and normal carotid artery were compared using one-way ANOVA tests  for continuous variables or chi-square tests for categorical variables, accordingly.We used restricted cubic splines  with fiv2), current smoker (no vs. yes), alcohol drinker (no vs. yes), fasting glucose (<7.0 vs. ≥7.0 mmol/L), TC (<6.2 vs. ≥6.2 mmol/L), HDL-C (<1.0 vs. ≥1.0 mmol/L), TG (<2.3 vs. ≥2.3 mmol/L) and LDL-C (<4.1 vs. ≥4.1 mmol/L) by including interaction terms  into the logistic regression models.In additional exploratory analyses, possible modifications of the association of physical activity level (per SD increase) with abnormal carotid artery and CP were also assessed for the variables, including sex , age (<60 vs. ≥60 years), BMI (<24.0 vs. ≥24.0 kg/mhttp://www.R-project.org/) was used for all statistical analyses. A two-tailed P<0.05 was considered to be statistically significant in all analyses.R software, version 3.5.3  Table . OverallHowever, there was no significant association of sedentary leisure time with CIMT  . Consistently, when physical activity level was assessed as quartiles, compared with participants in quartile 1, a significantly lower risk of abnormal carotid artery and CP were found in those in quartile 2 , quartile 3  and quartile 4 . Similarly, the risk of abnormal carotid artery and CP decreased through the quartiles of physical activity level (Pfor trend<0.001)  and as quartiles Table .Pinteraction<0.001). Moreover, a significantly stronger inverse association between physical activity level (per SD increase) and CP was also observed in elder participants .Stratified analyses were performed to assess the association of physical activity level (as a continuous variable) with the risk of abnormal carotid artery and CP in various subgroups Fig. . A signiThe significantly inverse association of physical activity level with abnormal carotid artery and CP was observed in participants with lower TC (<6.2 mmol/L) or LDL-C (<4.1 mmol/L), while the relationships were attenuated or even diminished among groups with higher TC (≥6.2 mmol/L) or LDL-C (≥4.1 mmol/L), with the test for interaction of TC and LDL-C with physical activity level >0.05 Fig. .Pinteraction>0.05 for all these stratified variables)  Fig. .The current study demonstrated that physical activity level was inversely associated with CIMT in population with high risk for CVDs. Furthermore, we found that a higher level of physical activity was associated with a lower risk of abnormal carotid artery and CP. A number of previous cohort and cross-sectional studies , 24–29 hThere are several established and potential mechanisms which may explain the reduced risk of CAS due to physical activity. First, physical activity reduces insulin sensitivity. Studies have shown that physical activity can enhance insulin sensitivity by regulating the insulin signaling pathway and the effectiveness of fatty acids , and decMore importantly, we observed that the inverse association between physical activity level and abnormal carotid artery and CP was more stronger in participants with age ≥60. The studies by Boss et al.  consistePrevious cohort and cross-sectional studies showed inconsistent association of sedentary leisure time with CIMT, abnormal carotid artery and CP. A previous cohort study in 614 healthy men and women reported the proportion of sedentary time was directly associated with baseline common carotid artery (CCA) IMT ; ParsonsOur study had some potential limitations. First, the study is a cross-sectional design, making it difficult to draw a causal conclusion. Replication of these findings in RCT and additional researches is needed. Second, an additional limitation of the study is the sole use of self-reported physical activity collected using a questionnaire, rather than by a validated physical activity questionnaire or objectively measured physical activity by gold standard approach, such as accelerometer. Third, the excluded participants were older than the enrolled ones, which may underestimate the benefit of physical activity on CAS (Supplemental Table In population with high risk for CVDs, physical activity was inversely associated with CIMT, abnormal carotid artery and CP, particularly among the elders. Sedentary leisure time was not associated with CIMT, abnormal carotid artery and CP. These results suggested that physical activity is important for carotid vascular health, and perhaps especially in elder population.Additional file 1.