In modern society, cancer is one of the most relevant medical problems. It is important to search for promising plant raw materials whose extracts have strong antioxidant and anticancer effects. The aim of this study was to determine the composition of phenolic compounds in plant extracts, to evaluate their antioxidant and anticancer activity, and to find the correlations between those activities. Extracts of calendula, sage, bearberry, eucalyptus, yarrow, and apple were selected for the study. The phenolic compounds of these extracts were determined by the UPLC-ESI-MS/MS method and the antioxidant activity was evaluated in vitro by four different UV-VIS spectrophotometric methods . The anticancer activity of extracts was tested against melanoma IGR39, glioblastoma U-87, and triple-negative breast cancer MDA-MB-231 cell lines in vitro by MTT assay. The highest content of identified and quantified phenolic compounds was found in sage leaf extract and the lowest in ethanol eucalyptus leaf extract. The highest antioxidant activity was determined by all applied methods for the acetone eucalyptus leaf extract. The majority of extracts were mostly active against the melanoma IGR39 cell line, and possessed the lowest activity against the glioblastoma U-87 cell line. Acetone extract of eucalyptus leaf samples exhibited the highest anticancer activity against all tested cell lines. Strong and reliable correlation has been found between antioxidant and anticancer activity in breast cancer and glioblastoma cell lines, especially when evaluating antioxidant activity by the FRAP method. Numerous scientific data confirm the benefits of natural antioxidants for human health . A relatEvaluation of the antioxidant activity of plant extracts is extremely important. It provides a scientific basis for the use of herbal preparations for the prevention and treatment of oxidative stress disorders. Herbal extracts have a variety of compounds that act as antioxidants through different reaction mechanisms. Therefore, the scientific literature notes that performing a single test of antioxidant activity is not adequate, thus it is recommended to use at least two different methods . Hence, ABTS and CUPRAC methods allow to assess the antioxidant activity of both hydrophilic and lipophilic antioxidants, and their pH is close to that of body fluids ,13. An aHerbal extracts are multicomponent matrices that can contain thousands of different biologically active compounds that have a wide variety of biological effects on the human body. The search for promising plant-based raw materials and analysis of their chemical composition and biological activity is particularly important when developing new drugs for the prevention and treatment of various diseases. For this reason, it is important to assess de novo the chemical composition of herbal extracts used in traditional and folk medicine using modern research methods, in order to determine which raw materials have the highest antioxidant, anticancer, and other biological activity in vitro and in vivo. Extracts of plant raw materials for this study  were selected owing to their strong antioxidant and anticancer effects. González-Burgos et al. indicate that eucalyptus leaf extracts have strong antioxidant and neuroprotective effects . Sage leOne of the most important bioactive compounds accumulated in plants is phenolic compounds, which are classified as natural antioxidants. Oxidative stress causes changes in cellular metabolism associated with DNA, protein damage, and lipid peroxidation . These cCancer is one of the most relevant societal, scientific, and medical issues in modern society, as morbidity and mortality are constantly increasing throughout the world . AlthougThe relationship between the antioxidant effect of plant extracts and their anticancer activity is controversial ,32. TherThe main aim of this study is to determine the composition of phenolic compounds in selected plant extracts in order to evaluate their antioxidant and anticancer activity and correlation between the estimates of these parameters. The obtained research results will provide new knowledge about the qualitative and quantitative composition as well as antioxidant and anticancer activity of selected plant extracts widely used in traditional and folk medicine and their correlation. This up-to-date information will be valuable from a theoretical and practical point of view to identify plant extracts with the strongest antioxidant and anticancer activity that could be promising for further in vivo studies, the manufacture of pharmaceuticals for the prophylaxis and treatment of cancer, and their use in clinical practice.Eucalyptus globulus Labill. and Salvia officinalis L. leaves, Achilleamillefolium L. herb, and Calendulaofficinalis L. flowers  were bought in the local pharmacy. Dried material was ground to a powder using a mill . The apples samples (cultivar ‘Ligol’) were supplied by the Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai, Lithuania . Each apple was cut into slices of equal size (up to 1 cm in thickness), and the stalks and seeds were removed. The slices were immediately frozen in a freezer (at −35 °C) with air circulation and then lyophilized with a Zirbus sublimator  at the pressure of 0.01 mbar . The lyophilized slices were ground to fine powder using a Retsch 200 mill . Loss on drying before analysis was determined by drying about 1 g of milled leaves in a moisture analyzer  to complete evaporation of water and volatile compounds (drying temperature: 105 °C).2. All cell cultures routinely were grown to 70% confluence and trypsinized with 0.125% TrypLE™ Express solution before passage. They were used until passage 20.Human melanoma cell line IGR39, human breast adenocarcinoma cell line MDA-MB-231, and human glioblastoma cell line U-87 ) were grown in DMEM Glutamax medium supplemented with 10% FBS and 1% antibiotics at 37 °C in a humidified atmosphere containing 5% CO•) radical, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium persulfate, ammonium acetate, sodium acetate trihydrate, iron (III) chloride hexahydrate, and 2,4,6-tripyridyl-s-triazine (TPTZ) were obtained from Sigma-Aldrich . Neocuproine was purchased from Alfa Aesar  and copper (II) chloride—from Carl Roth Carl Roth GmbH + Co. KG . Folin–Ciocalteu reagent, gallic acid monohydrate, sodium carbonate, aluminum chloride hexahydrate, and hexamethylenetetramine were purchased from Sigma-Aldrich GmbH . All analytical standards for UPLC-ESI-MS/MS analysis were HPLC grade and were also purchased from Sigma–Aldrich , except for and isorhamnetin 3-O-glucoside, which was from Extrasynthese . Deionized water, produced by the Milli–Q®  high-performance liquid chromatography water purification system, was used.All chemical solvents, reagents, and standards used were of analytical grade. Acetonitrile, acetone, and formic acid were obtained from Sigma-Aldrich GmbH  and ethanol from JSC Vilniaus degtinė . 1,1-Diphenyl-2-picrylhydrazyl  was obtained from Sigma-Aldrich . 3--2,5-diphenyltetrazolium bromide  was purchased from Sigma-Aldrich .TM Express, Dulbecco’s modified Eagle high glucose medium (DMEM Glutamax), fetal bovine serum (FBS), penicillin/streptomycin solution (100×), and phosphate buffered saline (PBS) were obtained from Gibco.All cell culture plastic ware was purchased from Thermo Fisher Scientific, Corning and Techno Plastic Products. TrypLEE. globulus and S. officinalis leaves, A. millefolium herb, and C. officinalis flowers was extracted as described by González-Burgos et al. . A volume of 3 mL of ABTS•+ solution (absorbance 0.800 ± 0.02) was mixed with 10 μL of the ethanol extract of apple leaves. A decrease in absorbance was measured at a wavelength of 734 nm after keeping the samples for 30 min in the dark. The regression equation for this assay was established to be y = 0.000084x − 0.002068; R2 = 0.998777.ABTS• free radical scavenging assay. The DPPH• free radical scavenging activity was determined using the method proposed by Brand-Williams et al. [• solution in 96.3% v/v ethanol  was mixed with 10 μL of the ethanol extract of apple leaves. A decrease in absorbance was determined at a wavelength of 517 nm after keeping the samples for 30 min in the dark. The regression equation for this assay was established to be y = 0.000111x − 0.000709; R2 = 0.998024.DPPHs et al. . DPPH• sy = 0.000048x − 0.001273; R2 = 0.998580 [CUPRAC assay. The CUPRAC solution included copper (II) chloride (0.01 M in water), ammonium acetate buffer solution , and neocuproine (0.0075 M in ethanol) (ratio 1:1:1). Here, 3 mL of CUPRAC reagent was mixed with 10 μL of extracts. An increase in absorbance was recorded at λ = 450 nm. The regression equation for this assay was established to be 3 × 6H2O (0.02 M in water), and acetate buffer  at the ratio of 1:1:10. A volume of 3 mL of a freshly prepared FRAP reagent was mixed with 10 μL of the apple leaf extract. An increase in absorbance was recorded after 30 min at a wavelength of 593 nm. The regression equation for this assay was established to be y = 0.000128x − 0.034745; R2 = 0.998682.FRAP assay. The ferric reducing antioxidant power (FRAP) assay was carried out as described by Benzie and Strain (11).The working FRAP solution included TPTZ (0.01 M dissolved in 0.04 M HCl), FeClTE) per gram of absolutely dry weight (DW). TE was calculated according to the following formula:c—the concentration of Trolox established from the calibration curve (in μM); V—the volume of leaf extract (in L); m—the weight (precise) of lyophilized leaf powder (in g).Calculation of antioxidant activity of the extract. The antioxidant activity of extracts was calculated from the Trolox calibration curve and expressed as μmol Trolox equivalent  and incubated at 37 °C for 24 h. Then, serial double dilutions of tested extracts (from 10 mg/mL to 0.156 mg/mL) were made in microplates. Cells treated only with medium containing 1.0% of ethanol served as a negative control. Free medium without cells was used as a positive control. After 72 h incubating at 37 °C, the cell growth medium in all the wells was replaced with the new one containing 0.5 mg/mL of MTT. After 4 h, the liquid was aspirated from the wells and discarded. Formazan crystals were dissolved in 100 μL of DMSO, and absorbance was measured at a test wavelength of 490 nm and a reference wavelength of 630 nm using a multi-detection microplate reader. The experiments were repeated three times independently and the results were given as means ± SD.EC50) values, reducing cell viability by 50%, were calculated.Applying Hill fit to compound dose–cell metabolic activity (absorbance) curves, the effective concentration  or −0.99–(−0.90) (negative); strong when r = 0.70–0.89 (positive) or −0.89–(−0.70) (negative); and moderate when r = 0.40–0.69 (positive) or −0.69–(−0.40) (negative).All the data obtained from this study were analysed statistically using SPSS™ software for Windows, Version 20.0  and the Microsoft Excel software package . Data are presented as mean ± standard error (S.E.) of at least three independent experiments. The correlation between antioxidant and anticancer activity was assessed by calculating Pearson‘s coefficient Qualitative and quantitative phenolic composition of tested extracts was investigated using the UPLC–ESI–MS/MS method .The total amount of identified and quantified phenolic compounds in the tested extracts ranged from 151.79 ± 6.21 mg/g  up to 1931.95 ± 80.37 mg/g (sage leaf extract). The total amount of phenolic compounds in eucalyptus leaf samples extracted with different extraction solvents (acetone and ethanol) did not differ statistically significantly and was significantly lower than that of other plant raw material extracts ; the extThe UPLC–ESI–MS/MS analysis of selected plant extracts showed that flavonoid compounds were the major component among all identified phenolic compounds, and 17 different flavonoids were identified and quantified. The total amount of flavonoids detected in the extracts of the analyzed samples varied significantly. The highest total amount of the identified and quantified compounds of the flavonoid group was found in marigold blossom extracts (1043.78 ± 47.5 mg/g), and the lowest in sage leaf extracts (33.52 ± 1.42 mg/g).The most abundant group of flavonoids in the investigated extracts is quercetin derivatives. The scientific literature indicates that quercetin and its glycosides have a broad biological activity. They might reduce the risk of cardiovascular diseases , metabolAnother group of phenolic compounds found in tested plant extracts was phenolic acids. The vast majority of the identified phenolic acids were hydroxycinnamic acid derivatives. Only one derivative of hydroxybenzoic acid, syringic acid, was identified. The highest total amount of identified and quantified phenolic acids (1898.43 ± 85.21 mg/g) was found in sage leaf extracts, and the lowest (81.93 ± 3.12 mg/g) in ethanol eucalyptus leaf extracts. Chlorogenic acid was predominant in most of the plant material sample extracts studied for a wide range of potential health benefits, including its strong antioxidant , anti-caQuinic acid (cyclitol), which is not classified as phenolic compound, was identified and quantified in the tested extracts. This acid was predominantly found in most of the tested plant raw materials, except for marigold and sage leaf extracts .Acetone extract of eucalyptus leaf samples exhibited the strongest antiradical activity assessed by the ABTS and DPPH methods  and reducing activity assessed by the CUPRAC and FRAP methods . The lowest antiradical and reducing activity in vitro, as assessed by all methods applied, was determined by examination of apple sample extracts; The differences in the results obtained may have been influenced by a variety of factors—different media or pH , differeThe antioxidant activity of plant extracts is determined by a complex of biologically active compounds. The scientific literature indicates that one of the strongest antioxidants that can determine the anti-radical and reducing properties of extracts is phenolic compounds . However•+ radical cation or DPPH• free radical [In order to evaluate the correlation strength between the in vitro antioxidant activities of tested plant extracts by different methods, the Pearson correlation coefficient was calculated . A stron radical ,9. The w radical ,11. The  radical . Various radical . The ant radical . As the  radical . The ant radical ,41. PerhThe effect of the tested extracts on cancer cell viability was variable . Six outThe majority of extracts were mostly active against the melanoma IGR39 cell line, and possessed the lowest activity against the glioblastoma U-87 cell line. However, this cell line is constitutively resistant to many chemotherapeutic agents owing to over-expression of P-glycoprotein and its stemness properties . Only onAcetone extract of eucalyptus leaf samples exhibited the highest anticancer activity against melanoma, triple-negative breast cancer, and glioblastoma cell lines . These results are consistent with the antioxidant activity, as this extract showed the highest antioxidant effect by all the methods used . The lowThe most active extract, made from eucalyptus leaves with acetone, possessed different activity on tested cell lines . The higHowever, contrary to expectations from antioxidant activity assays, ethanol extract of eucalyptus leaves did not exhibit high anticancer activity. In contrast to other eucalyptus leaf extracts made with acetone, this extract was from 2.4 to 9.5 times less active against different cell lines. These results can be explained in that the anticancer effect is not necessarily related to antioxidant properties of active substances, and such a phenomenon was observed previously with different substances with antioxidant activity in vitro . In our Apigenin, caffeic acid, catechin, chlorogenic acid, quercetin, kaempherol, and other compounds that are widely known to possess activity against cancer cells ,57,58,59Our results showed that anticancer activity could be related to antioxidant activity of extracts .A strong and reliable correlation was found between antioxidant and anticancer activity in breast cancer and glioblastoma cell lines, especially when evaluating antioxidant activity by the FRAP method. The same correlation between antioxidant and anticancer activity in melanoma cells was established as moderate; however, it was not found to be reliable. This could mean that the mechanism of anticancer activity in melanoma cells could be different and is less related to the scavenging free radicals or the antioxidant reducing activities.The extracts of calendula, sage, bearberry, eucalyptus, yarrow, and apple widely used in traditional and fold medicine contain 23 individual phenolic compounds, including 17 flavonoids. The highest amount of identified and quantified compounds was found in sage leaf extract. Rosmarinic acid was predominant in these extracts, while chlorogenic acid was predominant in the extracts of yarrow grass and apple tree fruits. Quinic acid, a non-phenolic compound, was identified and quantified and was found in the highest amount in the extracts of bearberry leaves.The highest antioxidant activity in vitro was determined using all applied methods in acetone extracts of eucalyptus leaves. The weakest antiradical and reductive activity in vitro by all methods was detected in apple extracts.Six out of eight extracts possessed cytotoxic effect against triple-negative breast cancer (MDA-MB-231), melanoma (IGR39), and glioblastoma (U-87) cell lines. Acetone extract of eucalyptus leaf samples exhibited the highest anticancer activity against all tested cell lines. A strong and reliable correlation was found between antioxidant and anticancer activity in breast cancer and glioblastoma cell lines, especially when evaluating antioxidant activity by the FRAP method.


To investigate the outcomes of primary squamous cell carcinoma (PSCC) of the breast undergoing radical surgery with or without adjuvant radiotherapy (RT).  A population cohort with histologically diagnosed PSCC of the breast was identified from the SEER database. The Kaplan–Meier method and Cox-regression proportional hazards model was used to assess the impact of surgical types with or without adjuvant RT on the cause-specific survival (CSS) and overall survival (OS). A retrospective analysis of PSCC between Jan 2010 and Dec 2018 from our institute was performed. p = 0.028) and OS (p = 0.048). Patients treated with LUMP + RT had better survival rates than patients who underwent lumpectomy , MAST alone , and MAST + RT , while postmastectomy RT did not significantly improve OS (p = 0.062) and CSS (p = 0.67) when compared to MAST alone. In addition, a total of 28 patients with PSCC of the breast were identified from our institute. All of these patients presented with estrogen receptor-negative type, and three of them had HER-2-positive PSCC; the median tumor size was 3 cm (range: 0.5–15 cm). Eight patients were treated with LUMP + adjuvant RT, thirteen with MAST, and seven with MAST + RT. Until the last follow-up of Sep 2021, 26 patients with PSCC were still alive and free of breast cancer, excepting that one patient treated with MAST and one patient with MAST + RT died from breast cancer.  A total of 515 patients with PSCC of the breast were included, 254 patients treated with mastectomy (MAST) alone, 78 with MAST + RT, 87 with lumpectomy (LUMP) alone, and 96 with LUMP + RT. The median follow-up time was 118 months (range: 0–379 months). In the multivariate Cox analyses, LUMP + adjuvant RT was an independent prognostic factor for CSS ( PSCC of the breast after radical surgery has a poor prognosis. Adjuvant RT after LUMP significantly improves survival of patients with PSCC of the breast. Further studies are still needed to investigate the role of adjuvant RT in PSCC of the breast after mastectomy. Primary squamous cell carcinoma (PSCC) of the breast is an epithelial type of metaplastic carcinoma according to the World Health Organization (WHO) classification of breast cancer . The diaDue to lack of adequate and accurate data, appropriate locoregional management, including surgical types or adjuvant radiotherapy (RT), for PSCC remains controversial, although most published studies have suggested that PSCC of the breast should be managed as invasive ductal carcinoma. As most patients present with large tumors, mastectomy (MAST) has been the most common surgical procedures for PSCC of the breast, although no data exist excluding breast-conserving surgery as an option , 10. In The present analysis is performed based on the SEER research database. The duration of the study was set from 1973 to December 2016, which was the record cutoff of the database. We obtained permission to access the data files from the SEER program by NCI with the reference number 11564-Nov 2019. As patient data identified from the database were deidentified and available to the public for research purposes, the ethical approval of the present study was waived by the local ethics committee.In addition, a retrospective analysis of SCC of the breast cancer between 2010 and 2018 was performed in Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, and a total of 28 cases were identified.Among the 28 included patients, eight patients were treated with LUMP + adjuvant RT, 13 with MAST, and 7 with MAST + RT. Fifteen patients underwent axillary lymph node dissection, and seven patients underwent sentinel lymph node biopsy (sentinel lymph node negative). Axillary lymph node dissection was not performed on two patients. Chemotherapy was administered to twenty-five patients, and adjuvant radiotherapy was administered to fifteen patients. Hormonal therapy was administered to one patient presented with low PR positivity (2%).∗ Stat 8.3.5 software.All cases assigned the ICD-0–3 histology coded as 8070/3, 8071/3, 8072/3, 8073/3, 8074/3, 8075/3, 8076/3, 8077/3, and 8078/3 for primary tumor site of the breast were included in the present analysis. Age, race, sex, surgery, tumor size, TNM stage, number of positive LN, chemotherapy, radiotherapy, and sequence, grade, ER status, PR status, and year of diagnosis were abstracted from the SEER database. All the patients were categorized by the 6th edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) tumor-node-metastasis (TNM) staging system. Age was also converted to a binary data point using a cutoff of 50 years (≤ 50 vs. > 50 years). Race was classified as white or nonwhite. Surgery was classified as mastectomy (MAST) or lumpectomy (LUMP). Those with unknown surgery or no surgery status were excluded from analysis. Adjuvant radiotherapy was classified into yes or no. Therefore, locoregional treatment regimens were then divided into the following four groups: LUMP, LUMP + RT, MAST, and MAST + RT. The primary tumor site of the breast was determined via the ICD-0–3 site code. Adjuvant chemotherapy was classified into yes or no/unknown. Data for some analyzed variables  were incomplete. Those without data were excluded from statistical analysis involving those variables. We extracted the data using case listing session of SEER p < 0.1 in univariate analysis were put into the multivariate analysis. The statistical analysis was performed using the software of NCSS 11 Statistical Software (2016) . The statistical difference was considered as significant when p < 0.05.In the SEER database, survival is calculated as the number of months from cancer diagnosis to the date of death. For the estimation of cancer-specific survival (CSS), women who died from causes other than PSCC of the breast were censored. The 10-year CSS and OS were calculated using the Kaplan–Meier method. Baseline clinicopathological characteristics were compared between the treatment groups by using chi-square and Fisher's exact probability tests. Baseline clinical variables were included in the univariate and multivariate survival analyses. The variables which had Between 1973 and 2016, a total of 515 patients with PSCC of the breast were identified from the SEER database. In the entire cohort, all of the patients were female and treated with radical surgery. Approximately 83.9% of them were white, and 16.1% were nonwhite. The median age of the PSCC of breast was 66 years, with 81.8% of the patients older than 50 years. The median tumor size was 3.55 cm (range: 0.2–52 cm). As for the local treatment strategies for PSCC of the breast, 254 patients (49.3%) were treated with mastectomy (MAST) alone, 78 (15.1%) with MAST + RT, 87 (16.9%) with lumpectomy (LUMP) alone, and 96 (18.6) with LUMP + RT. Approximately, 41.7% of them received adjuvant chemotherapy. In consistent with previous reports, the expression of hormonal receptor in PSCC of the breast was relatively low and 19.0% (71/374) and 11.3% (42/327) of patients were positive for ER and PR, respectively. In addition, half of the included patients (56.6%) presented with stage II (243/429), followed by stage I (22.1%) and stage III (21.2%). The baseline characteristics are listed in p = 0.0006, p < 0.001). However, no significant CSS difference was observed between the MAST group and MAST + RT group (p = 0.67). In addition, the 10-year OS for patients treated with LUMP + RT, LUMP, MAST, and MAST + RT was 72%, 47%, 53%, and 40%, respectively . In addition, adjuvant RT after mastectomy among PSCC patients had a tendency to improve OS (p = 0.06). Furthermore, it should be noted that the prognosis of PCSS who were treated with mastectomy ± adjuvant RT was poorer than that treated with LUMP ± adjuvant RT. One possible explanation for this finding was that the tumor characteristics of PSCC patients in the mastectomy arm are more aggressive; thus, the prognosis of this patient cohort in mastectomy will be slightly worse than that in the LUMP group. As shown in The median follow-up time was 118 months (range: 0–379 months). A total of 122 patients died of breast cancer. The 10-year CSS and OS were 77% and 49%, respectively. For the Kaplan–Meier analysis, the 10-year CSS was 93%, 81%, 73%, and 66% for patients treated with LUMP + RT, LUMP, MAST, and MAST + RT, respectively (p = 0.061), tumor stage , nodal status , and treatment regimen  were significantly related to CSS of PSCC. Given the limitations of univariate analysis, multivariable Cox analysis was performed to investigate the independent factors associated with CSS (p = 0.01) was an independent predictor for worse CSS, while the prognosis of PSCC patients treated with LUMP + adjuvant RT was significantly better than that in patients treated with LUMP alone , stage , T stage , nodal status , PR status , and treatment regimens  were significantly related to OS of PSCC. Multivariate analysis demonstrated that age, PR status, chemotherapy, T stage, nodal status, and treatment regimens were independent factors for OS among PSCC patients  and OS (p < 0.001) in comparison with BCS alone. We also find that adjuvant RT after mastectomy among PSCC patients has a tendency to improve OS (p = 0.06), but not for CSS (p = 0.67). Multivariate Cox analysis indicates that the treatment regimen is an independent predictor for OS and CSS. However, data on cancer recurrence could not be made available from the SEER database, and thus, we were unable to compare the locoregional recurrence rate in PSCC of the breast treated with or without radiotherapy. Based on our findings, adjuvant RT should be recommended for patients with PSCC of the breast after breast-conserving surgery. And, LUMP + RT could be an alternative treatment option for early-stage PSCC of the breast. But, the role of adjuvant RT after mastectomy is still needed to be investigated in further studies.A total of 515 patients with PSCC of the breast are finally included for analysis in the present study. In consistent with previous studies, most cases of PSCC of the breast occur in elderly patients (81.9% older than 50 years), with a median age of 66 years. In addition, PSCC of the breast has low ER or PR positivity (19.0% in ER positivity and 11.4% in PR positivity) and high histologic grade (59.5% in poor or undifferentiated). All of these features indicate that PSCC of the breast has a poor prognosis. Indeed, the 10-year OS of PSCC of the breast in the present study is 49%, which is significantly lower than the prognosis of invasive breast cancer  . TherefoInconsistent with findings from SEER data, 23 patients (82.1%) were older than 50 years with a median age of 54.5 years. In addition, all of these patients presented with hormonal receptor-negative type, excepting for one patient with low PR positivity (2%) and three of them with HER-2-positive PSCC. Two patients with HER-2-positive PSCC were treated with trastuzumab without disease recurrence. As for the survival outcomes of PSCC in our cohort, 26 patients with PSCC were still alive and free of breast cancer, excepting that one patient treated with MAST and one patient treated with MAST + RT died from breast cancer. Due to the limited sample size, whether LUMP + RT would be superior to MAST alone for PSCC of the breast remains undetermined in further studies.So far, no adjuvant chemotherapy regimens for PSCC of the breast were established. Published studies indicated that PSCC of the breast was resistant to standard chemotherapy  for invasive breast cancers. Therefore, some researchers proposed the use of a cisplatinum-based chemotherapeutic regimen, which is commonly used for squamous cell carcinoma in the other sites. In our study, we find that adjuvant chemotherapy is an independent predictor for OS, but not for BCSS. However, the specific chemotherapy regimens used could not be available from the SEER database. In our cohort data, 25 PSCC patients received adjuvant chemotherapy. Of them, fifteen patients (60%) were treated with epirubicin (E) and cyclophosphamide (C) followed by docetaxel (T) (EC-T), four patients were treated with carboplatin-based chemotherapeutic regimen, two patients were treated with cyclophosphamide and docetaxel, one patient was treated with epirubicin (E) and cyclophosphamide (C) followed by paclitaxel (p), one patient was treated with epirubicin, cyclophosphamide, and 5-FU, one patient was treated with capecitabine alone, and the remaining one patient with unknown chemotherapy regimen.Our study still has several limitations. First of all, selection bias existed among the four groups. For example, more patients with stage III receives MAST alone (51.6%) or MAST + RT (33%), when compared to LUMP alone (5.5%) and LUMP + RT (9.9%). Due to the rarity of this disease, we could not perform the PSM method to decrease the bias resulting from unevenly distributed measured covariates. Secondly, there is a sizeable amount of missing data regarding tumor stage and hormonal receptor status in the SEER database, which could be another source of bias. Thirdly, according to NCCN guideline, omitting radiotherapy in SCC patients undergoing lumpectomy remains unknown, especially for those with poor histology and who are young age. In our study, a total of 87 SCC patients were treated with LUMP alone. Of them, 17 patients were younger than 50 years. In addition, whether adjuvant radiotherapy should be performed for patients with stages I and II undergoing mastectomy remains undetermined, especially for those with negative lymph metastasis. Both of them would impact disease recurrence and survival. Finally, disease recurrence is not obtained from the SEER database. Therefore, we are unable to determine recurrence patterns and investigate factors associated with disease-free survival and locoregional relapse-free survival.In conclusion, our findings show that the prognosis of PSCC after radical surgery remains poor. Adjuvant RT after BCS significantly improves survival when compared to BCS alone. Further studies are still needed to investigate the role of adjuvant RT in PSCC after mastectomy.


Considering the involvement of genetics in migraine pathogenesis in diverse ethnic populations, genome-wide association studies (GWAS) are being conducted to identify migraine-susceptibility genes. However, limited surveys have focused on the onset age of migraine (AoM) in Asians. Therefore, in this study, we aimed to identify the susceptibility loci of migraine considering the AoM in an Asian population.We conducted a GWAS in 715 patients with migraine of Han Chinese ethnicity, residing in Taiwan, to identify the susceptibility genes associated with AoM. Based on our standard demographic questionnaire, the population was grouped into different subsets. Single-nucleotide polymorphism (SNP) associations were examined using PLINK in different AoM onset groups.ESRRG was associated with AoM CUX1, rs146778855 in CDH18, rs117608715 in NOL3, rs150592309 in PRAP1, and rs181024055 in NRAP were associated with the later AoM.We discovered eight novel susceptibility loci correlated with AoM that reached the GWAS significance level in the Han Chinese population. First, rs146094041 in To our knowledge, this is the first GWAS to investigate the AoM in an Asian Han Chinese population. Our newly discovered susceptibility genes may have prospective associations with migraine pathogenesis.The online version contains supplementary material available at 10.1186/s10194-021-01301-y. Migraine is the most common debilitating neurological disorder, presenting repeated moderate to severe, throbbing, and unilateral headaches, which may also be accompanied by nausea, vomiting, photophobia, or phonophobia , 2. ApprThe estimated life-time prevalence of migraine is 15–20 % . AccordiThe prevalence rate of migraine is 4–11 % in children aged 7–11 years in the United States . Early iPrevious studies have shown aggregation in families as a feature of migraine, highlighting its causal genetic architecture. Gormley et al. applied migraine polygenic risk scores (PRS) to investigate the common genetic variant load in 8,319 participants from 1,589 families in Finland . The resTherefore, the aim of this study was to identify the susceptibility loci of migraine considering the age of migraine onset (AoM) in an Asian population. We hypothesized that some genetic factors may be associated with the AoM. This cohort study involved two-stage clinic-recruitment of migraine participants. We stratified the patients by age groups ≤ 12 years old versus > 12 years old and established a linear regression model for AoM in all cohorts. We aimed to discover susceptibility single-nucleotide polymorphisms (SNPs) associated with the AoM with and without aura in a Han Chinese population residing in Taiwan.This study involved a cohort of 715 cases of migraine with and without aura. The study protocol was approved by the Tri-Service General Hospital (TSGH) Institutional Review Board. All individuals provided signed, informed consent before enrollment. Each participant completed a screening questionnaire and was later interviewed by a board-verified neurologist and headache consultant (FCY). The diagnosis of migraine was in accordance with the criteria in the third edition of the International Classification of Headache Disorders (ICHD-III) . The parAll participants completed a uniform questionnaire, encompassing demographic characteristics, AoM, Migraine Disability Assessment (MIDAS) , 16, famThe prevalence of migraine is similar between boys and girls in the prepubertal stage . Howeverhttps://www.twbiobank.org.tw/new_web/exp_doc/TWBv2.0_SNPs%E4 %BD%8D%E9 %BB%9E%E7 %9B%B8 %E9 %97 %9 C%E8 %B3 %87 %E8 %A8 %8 A.zip. Of these SNPs, approximately 446,000 SNPs were associated with the Taiwanese genotypic background, 105,000 SNPs were clinically significant, and others are associated with disease, drug response, and metabolism, as determined by Thermo Fisher Scientific over the years. The signal CEL files generated from the Axiom TWB 2.0 SNP array were transformed to genotyping data (tped and tfam) using Analysis Power Tools.Blood samples from patients were collected in 5-ml EDTA vacutainers . Genomic DNA was extracted from the blood samples using the QIAamp DSP DNA Mini Kit on the QIAsymphony platform . DNA quality was measured using a NanoDrop One spectrophotometer . The genomic DNA was then applied to Affymetrix Axiom Genome-Wide TWB 2.0 arrays, which included approximately 752,921 probes for 686,463 SNPs . The comP < 0.00001). The platform of genotyping in NCGM is tested annually with thousands of samples to confirm the accuracy of genotyping to correct the standard criteria for all genotyping experiments. Detailed information of Taiwan Biobank is presented on its official website (https://taiwanview.twbiobank.org.tw/index).Quality control procedures were conducted using TWB2 SNP criteria and PLINK. All data of DNA quality were tested and recorded, such as OD260/230 > 1.0 and OD260/280 > 1.8, and the concentration > 15 ng/µl. Before genotyping, we eliminated the missing metadata from typed files by clinical metadata for phenotype association study. After gathering all detected loci in arrays, we evaluated the quality of genotyping based on dish quality control (DQC) value > 0.82, average call rate for passing samples > 98.5 %, average genotype calling rates < 97 %, and the Hardy-Weinberg equilibrium (P < 5 E-08 (with *) and were annotated using the NCBI based on the RefSeq database [https://gtexprotal.org) by downloading 50 tissue cis-expression quantitative trait locus (cis-eQTL) data with FDR cut-off 5 % to identify the relationship of variant-gene regulation [Based on our standard demographic questionnaire, the population was grouped into different subsets. SNP associations were examined using PLINK based on the groups for AoM. For the first stratification, in the phenotype association study, we examined the variant relationship between groups with the AoM onset before and after 12 years using the 1df chi-square allelic test, and another quantitative trait association study was applied to determine two different distributions in all migraine cohorts using regression statistics and the Wald test. In the second stratification, all original groups were separated based on the presence and absence of aura and were tested using the quantitative trait association study. Finally, the significant variants were retrieved with database  with ANNdatabase . To elimdatabase . In addigulation .P < 0.05). Migraineurs with aura had earlier AoM and higher MIDAS scores than migraineurs without aura.Table ESRRG (P = 3.40 E-09), was more significant, with P < 5 E-8  has a more significant value than P < 5 E-8 in two different AoM distributions  and 1 SNP (rs150592309 in PRAP1) were almost significant in the aura groups . However, we found that the exonic variant rs181024055 in NRAP  significantly reached the threshold  was lower than that in the alternative genotype TC (approximately AoM = 48 years). We thus suggest that this variant is more associated in the cohorts of migraine without aura.We also compared the migraine groups without aura. In the before and after 12-year-old groups, no variants reached the threshold phenotypes and characteristics of migraine facilitate diagnosis, identification of pathology, and eventual discovery of targets for therapy. In this study, rs146094041 in ESRRG encodes estrogen-related receptor gamma (ERRγ), also known as nuclear receptor subfamily 3, group B, member 3 (NR3B3). ERRγ is an orphan receptor to a member of nuclear steroid hormone receptors. Estrogen signaling and estrogen-related receptors have been extensively associated with the pathogenesis of auditory function and migraine [ESRRG expression is highly upregulated in cochlear hair cells and that it is linked with a candidate gene for senile hearing impairment [ESRRG on chromosome 1 is involved in a phenotype including hearing impairment and delayed development [ESRRG and the mechanism of auditory symptoms as well as the environmental effects on migraine.migraine –26. For pairment . Schilitelopment . Moreoveelopment . Additioelopment . These fNRAP encodes nebulin-related anchoring protein, a muscle-specific isoform belonging to the nebulin protein family. NRAP is associated with the link between mature muscle cells and myofibrillar myogenesis during growth. NRAP mutation is associated with dilated cardiomyopathy [NRAP in migraine pathogenesis has not been described. Thus, NRAP may have a novel role in migraine pathogenesis.myopathy , 32. TheCUX1 encodes cut like homeobox 1 (Cux1), a homeobox transcription factor that is expressed ubiquitously in all mammalian tissues. Cux1 is involved in regulating gene expression, cell cycle progression, morphogenesis, and differentiation [Kip1 [CUX1 is functionally involved in migraine.ntiation . In the ntiation . Li et aon [Kip1 . Cubeloson [Kip1 . Furtheron [Kip1 . OverallCDH18 encodes cadherin 18, a type II classical cadherin that mediates calcium-dependent cell-cell adhesion. CDH18 is expressed particularly in the brain and is involved in regulating neural morphogenesis, including the formation and plasticity of synapses [CDH18 are associated with autism spectrum disorders [CDH18 gene show a robust association with depression [CDH18 on the mechanisms involved in migraine.synapses . Structuisorders . Additiopression . OverallNOL3 encodes nucleolar protein 3 (NOL3) in humans. NOL3 is highly expressed in several organs including the skeletal muscle, heart, and the brain. NOL3 has a functional CARD domain that acts as an apoptosis repressor by downregulating caspase-2 and caspase − 8 activities [NOL3. Further in vitro and in vivo experiments verified that NOL3 mutation alters the post-translational modification of NOL3 and that neuronal hyperexcitability is absent in Nol3 knockout mice [NOL3 mutation may also cause delayed AoM by a loss or decrease-of-function in neuronal excitability, although the exact pathogenesis associating NOL3 and migraine needs further investigation.tivities , 41. Jonout mice . NOL3 muThis study has several strengths. First, all participants underwent a structured interview through authenticated questionnaires. Thus, we cautiously excluded cases of other types of headaches, particularly tension type and medication overuse headaches, which could alter the clinical features. Second, the genes associated with AoM onset in this study are mostly expressed in the nervous system and are involved in neuronal excitability and neurogenesis. Third, migraine is associated with a high prevalence of psychiatric comorbidities including mood and autism spectral disorders. The potential mechanism of migraine comorbid mood disorders includes the dysregulation of the normal sensory network and alternation of functional connections between the brainstem-regulating circuits and the neuro-limbic centers , 43. NeuThis study also has some limitations. First, the sample size of this study is relatively small and may partially limit its statistical power. Enrolling more migraineurs, especially aura participants, is thus warranted to validate these results in the future. Second, the proportion of patients in the aura group in this study, 23.3 %, is higher than that in a previous Asian study 10 %). This could be because the participants were all recruited from Dr. Yang’s outpatient clinic focusing on headache. Dr. Yang is a headache specialist and he received referral of patients with intractable headache from clinics, especial migraine with aura because of its unique presentation and higher risk of comorbidity . This is0 %. ThisSeveral novel susceptibility genes associated with AoM, most of which were probably insinuated in migraine, were identified in this study. Thus, this study will enhance the diagnosis and treatment of patients with migraine; however, a larger sample is necessary to obtain the definitive proof.Additional file 1: Supplementary Table 1. Functional Clustering Analysis of genes of candidate variants


Briefly, black truffle extract was administered orally to treat streptozotocin- (STZ-) induced diabetic Wistar rats for 45 days. At the end of the experimental duration, rats were sacrificed to perform biochemical and gene expression analyses related to lipid regulatory and inflammatory pathways. Our results indicated that total cholesterol, triglycerides, free fatty acids, phospholipids, and low-density lipoprotein in different tissues and circulation were significantly increased in diabetic rats. Furthermore, the β-hydroxy β-methylglutaryl-CoA enzyme was also significantly increased; lipoprotein lipase and lecithin–cholesterol acyltransferase enzymes were significantly decreased in diabetic rats. However, the above conditions were reversed upon black truffle extract feeding. Furthermore, black truffle extract was also found to downregulate the expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-6) and lipid regulatory genes (serum regulatory element-binding protein-1 and fatty acid synthase). The truffle extract-treated effects were comparable to glibenclamide and medication commonly used to treat diabetes mellitus. Overall, our results suggested that black truffle possesses strong antihyperlipidemic and anti-inflammatory effects on diabetic rats. These findings will enhance the current knowledge about the therapeutic importance of black truffles. They might be exploited as a possible food supplement or even as a natural source of pharmaceutical agents for diabetes prevention and treatment.Black truffle, a culinary and medical fungus, is highly valued worldwide for its nutritional and therapeutic importance. To enhance the existing knowledge about the beneficial properties, this study investigates the antioxidant, antihyperlipidemic, and anti-inflammatory effects of black truffle extract in  Tuber melanosporum), a hypogenous and edible fungus, is considered delicious and cherished worldwide [Black truffle  is a metabolic disorder associated with various alterations due to either insufficient insulin or lack of action . MetabolκB) participates in the inflammatory response, resulting in activation of NF-κB1 (p50/p105) and Re1A (p65). This activation can efficiently induce the expression of proinflammatory cytokines , interleukin (IL)-6, IL-1 alpha), chemokines, adhesion molecules , and inflammatory enzymes  [DM patients exhibit a strong hyperglycemic condition in an oral glucose tolerance test (OGTT). Hyperglycemia is a well-known condition associated with inflammation. The inflammatory responses are controlled by initiating several signaling pathways that regulate the expressions of pro- and anti-inflammatory mediators . The pro, COX-2) .With context to this, we have evaluated the therapeutic effects of the black truffle by evaluating its bioactive components and how the presence of those active components treats diabetic rats. In addition, we have studied the antihyperlipidemic effects of black truffle extract via assessing lipid parameters and identified a relationship between inflammation and hyperlipidemia in diabetic conditions. The overall results can help us conclude the antihyperlipidemic and anti-inflammatory properties of black truffle extract and could be beneficial for food scientists and pharmacologists to develop functional food or source of new innovative drugs.T. melanosporum samples were collected from the wild environment of Chuxiong , the major black truffle-producing area in China. The samples were collected at the same ripening stage, color, and shape without physical damage. After identification, samples were washed to remove contaminants and transversely cut into slices, followed by drying at 40°C for 24 h. The dried truffle samples were ground into a fine powder (<0.425 mm) by a grinder to increase extraction efficiency and then stored in the dark at 4°C before analysis.Streptozotocin (STZ) and the standard antidiabetic drug glibenclamide (GB) were purchased from Sigma-Aldrich . All primers were designed using PubMed and obtained from Sangon Biotech, Guangzhou, China . TRIS, tThe sample powder was pretreated with 70% ethanol twice to remove small molecular compounds. Then, the deionized water was added to the sample powder in a ratio of 5 : 1, and the mixture was kept boiling for 2 hours. Finally, the extract was filtered and concentrated in a dry solid form using a rotatory evaporator .Male albino Wistar rats were used to induce DM. The rats were selected in a weight range of 180-220 g. The temperature and humidity of the rising condition were maintained at 25°C and 40%. Polypropylene cages were used for the rats living under a 12 h light-dark cycle with free food and water access. A standard pellet diet was purchased from Southern Medical University  . Rats were kept for two weeks as an acclimatization period before the actual experiment began. The complete protocol and the ethical guidelines for the usage of rats were approved  by the ethical conduct in the care and use of animals.After the acclimatization period, rats were induced experimental DM by STZ (citrate buffer was used to prepare the 0.1 M STZ) intraperitoneal injection at a dose of 40 mg/kg b.w. After three days, Roche Diagnostics plasma  was used to estimate the plasma glucose levels. Fasted rats with high glucose levels (>250 mg/dL) were considered diabetic and used for further study.n = 6), out of which 18 were diabetic rats and 12 were control rats. Solid black truffle extract was dissolved in deionized water to prepare the final black truffle extract of 400 mg/kg b.w. The dose was fixed according to our previous study [A total of 30 rats were divided into five groups Group 3: diabetic rats (STZ 40 mg/kg b.w.)Group 4: diabetic+black truffle extract (400 mg/kg b.w.)Group 5: diabetic+glibenclamide (600 μg/kg b.w.)At the end of the experiment, rats were kept in fasting conditions overnight and anesthetized with 24 mg/kg b.w. of ketamine hydrochloride to be sacrificed. A mixture of potassium oxalate and sodium fluoride in a ratio of 3 : 1 was added to the blood samples. The liver, kidney, and other related tissues were dissected and stored at -80°C for further lab analysis.v/v/v) each time. The combined extracts were centrifuged at 3000 rpm for 10 min, and the final volumes were recorded. The supernatants were used to determine TPC, TFC, and antioxidant activities. A UV-visible spectrophotometer measured absorbance at 765 nm by a UV-visible spectrophotometer . The TPC values were calculated by a calibration curve of gallic acid and expressed as milligram GAE per gram. The TFC values were calculated by a catechin calibration curve and expressed as milligram CAE per gram.The extract's total phenolic content (TPC) was determined using the Folin-Ciocalteu phenol reagent by the previously reported colorimetric method . The alu2+ equivalent per gram of the black truffle extract on a dry weight basis. Furthermore, a colorimetric method was carried out to determine metal chelating ability (MCA) based on a previously published procedure [The 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging capacity of black truffle extract was determined according to the method described by Miller et al.  Antioxidrocedure . The val4 was added into the tube and centrifuged at 6000 rpm for 30 min. Finally, the absorbance of the supernatant was measured at 280 nm by an ultraviolet spectrophotometer. In this assay, 25 mM Tris-HCl buffer (pH 7.4) was used as the blank. According to our previous research, aspirin was used as the positive control, and the IC50 was 1.94 mg/mL [50 . blank is the absorbance of the blank, Abssample is the absorbance of the sample, and IC50 can be calculated based on extract concentration and inhibition rate.The protease inhibitory assay was used with a slightly modified colorimetric assay according to Kumarappan et al.  Briefly,94 mg/mL . Assays v/v) solution was used to homogenize tissue and extract lipid from the tissue by following the method of Folch et al. [A classic method was used to extract the lipids from the tissues. Briefly, the cold saline was used to rinse the tissue, and then, a cold chloroform-methanol , lipoprotein lipase (LPL), and 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase was estimated as described by Hitz et al. , Korn 3, and PhiThe plasma CRP level of each rat was tested using the kit .α were electrophoresed on a 1.0% agarose gel, and ethidium bromide dye was used to visualize the bands. The band intensities were captured with an ImageQuant LAS 500  and quantified using ImageQuant analyzer software. GAPDH was used as a loading control for the comparison of obtained results.According to the manufacturer's procedure, total RNA was extracted using the RNeasy mini kit (50) . The cDNA synthesis kit  was used to synthesize the first strand of cDNA from the total RNA and amplify a polymerase chain reaction (PCR). The PCR cycle settings are as follows: [p < 0.05 are considered statistically significant.The statistical significance was analyzed using SPSS version 15 , one-way analysis of variance (ANOVA) was used, and to find individual comparisons, Duncan's multiple range test (DMRT) was used. All data were denoted as the mean ± standard deviation (SD). Values μmol TE/g, 41.01 μmol TE/g, 12.12 mmol Fe2+E/100 g, and 28.54 μmol EDTA E/g, respectively  is crucial in determining the antioxidant activity of truffle extract. The total phenolic and flavonoid contents were 11.24 mg GAE/g and 4.61 mg CAE/g, respectively. For the antioxidant capacities, the value of ABTS, DPPH, FRAP, and MCA was 88.03 ectively .50 value are shown in 50 value of black truffle extract was 0.92 mg/mL, and the IC50 of aspirin (the positive control) was 1.94 mg/mL.The protease inhibitory rate of 5 concentrations of black truffle extract and ICp < 0.05). Similar results were achieved in the rats treated with GB with no significant difference.Tables p < 0.05), comparable to that of control rats. The truffle extract-treated effects are similar to GB.α. The diabetic rats showed altered gene expressions, and the values were significantly (p < 0.05) recovered upon the treatment with truffle extract. Similar results were obtained in the group of rats treated with GB.α-glucosidases and treat diseases related to carbohydrate metabolisms, such as postprandial hyperglycemia and diabetes [in vitro and animal studies also explored the health-promoting properties of dietary flavonoids on glucose and lipid homeostasis and disease prevention, such as diabetes, cancers, cardiovascular disease, and osteoporosis [in vitro antioxidant black truffle extract activity was assayed via various studies such as ABTS, DPPH, FRAP, and MCA , total cholesterol (TC), very low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) . Other pHMG-CoA reductase can catalyze HMG-CoA to mevalonic acid, a critical rate-limiting step in cholesterol biosynthesis, and various statins exert beneficial effects through targeting this pathway . In our Since hyperlipidemia is strongly associated with DM, understanding the interrelationship between the insulin-signaling pathway and the lipid regulatory pathways becomes crucial . Hence, Fatty acid synthase (FAS) is an essential enzyme for synthesizing fatty acids, and further esterification allows them to store in adipose tissue . This acα and IL-6 [50 values for the in vitro anti-inflammatory study of black truffle extract are shown in α and IL-6 were increased in the diabetic rats in response to hyperlipidemia. Both expressions were reduced upon the treatment with truffle extract. C-reactive protein is an acute-phase protein found to be increased in inflammation. Our study found that CRP levels were increased in circulation in diabetic rats and reduced significantly upon the treatment with truffle extract. Similar results were achieved for diabetic rats treated with GB. The insulin-mediated lipid regulation may contribute to reducing inflammation in adipose tissue. It is widely accepted that insulin directly involves in modulating the inflammatory response and improving the immunocompetence in immune cells.In adipose tissue, hyperlipidemia is directly involved in releasing proinflammatory cytokines like TNF-and IL-6 . The IC5in vitro studies. Black truffle extract also regulates lipid homeostasis and lipid metabolizing enzymes and genes involved in fatty acid metabolisms such as SREBP-1C and FAS. The CRP and inflammatory cytokine (TNF- α and IL-6) mRNA studies show that black truffle extract regulates inflammation. The phenolic and flavonoid contents of the black truffle extract regulate various metabolic pathways. A complete analysis of its role in regulating other key pathways of diabetes mellitus could enable the availability of black truffle as an important antidiabetic agent shortly.Overall, black truffle extract exhibited a strong antioxidant activity, which was documented through


The anti-inflammatory effect of exclusive enteral nutrition on the gut of children with Crohn’s disease is rapidly lost after food reintroduction. This study assessed disease dietary triggers following successful treatment with exclusive enteral nutrition.Nutrient intake, dietary patterns and dietary biomarkers in faeces  were assessed in 14 children with Crohn’s disease during early food reintroduction, following exclusive enteral nutrition. Groups above (Group A) and below (Group B) the median levels of faecal calprotectin after food reintroduction were assigned for comparative analysis.p = 0.03; Red and processed meat: 151  vs. 63.3 , p = 0.02; gluten-containing cereals: 289  vs. 203 , p = 0.035). A diet consisting of cereals and meat products was predictive (92% accuracy) of higher faecal calprotectin levels after food reintroduction. In faeces, butyrate levels, expressed as absolute concentration and relative abundance, were higher in Group A than Group B by 28.4 µmol/g (p = 0.015) and 6.4% (p = 0.008), respectively. Levels of gluten immunogenic peptide and starch in faeces did not differ between the two groups.Intakes of fibre, gluten-containing cereals and red and processed meat were significantly higher in Group A than Group B; .The online version contains supplementary material available at 10.1186/s12876-021-02029-4. The increasing incidence of Crohn’s disease (CD) in societies in economic transition, suggests that environmental factors, including a Western diet are major contributors to the disease pathogenesis . In nutrExclusive enteral nutrition (EEN) is the only established dietary treatment for active CD in children , 6. In aIn the current study, we performed detailed assessment of the diet of children with CD during this early phase of food reintroduction, following successful EEN, and explored relationships with FC. We concentrated our efforts on this critical food reintroduction period, which provided us with a unique timeframe to explore dietary disease triggers in a population with homogeneous characteristics of disease activity, type, and duration of preceding EEN treatment at study enrolment.Children (aged 3–17 years) with a new diagnosis of CD or with disease in relapse, who initiated an 8-week course of EEN were recruited from the Royal Hospital for Children, Glasgow and neighbouring hospitals, as described previously . A faecaDisease activity was assessed with the weighted Paediatric Crohn’s Disease Activity Index (wPCDAI) . In the Participants recorded their diet for three days prior to faecal sample collection, including intake of enteral nutrition as maintenance treatment (MEN), with 3-day food diaries, estimating weight of consumed foods using household measures. MEN was prescribed to provide 20–25% of daily energy requirements. No specific food reintroduction guidance was provided to patients after EEN cessation. Dietary records were analysed using the WinDiets software  for calculation of energy and nutrient intake.Dietary patterns were assessed by classification of individual foods in groups based on the grouping applied in the UK National Dietary and Nutrition Survey (NDNS) . IndividThe entire bowel movement was collected within 4 h of defecation and was transferred to the laboratory on ice under anaerobic conditions . SamplesShort and branched chain fatty acids  were measured using gas chromatography (Agilent 7890A) , and werStatistical analysis was performed using Minitab, Version 18  and R (version 3.5.3). Patients were divided into two equally numbered groups based on their FC levels during food reintroduction. Patients with an FC above the median concentration after food reintroduction were assigned to Group A (Above) and patients with an FC below the median concentration to Group B (Below). Continuous data were expressed as medians with interquartile ranges  and categorical data as counts with frequencies (n [%]), unless otherwise stated. Comparisons of continuous variables between different groups were performed on Box-Cox transformed data using general linear models and Fisher’s least significance difference post-hoc test. Comparisons of dietary intake and biomarker levels between the two groups were adjusted for FC levels at EEN completion (p-adj). Relationships between dietary components and FC levels were explored with Pearson and Spearman rank correlations.leaps  days of food reintroduction. None had commenced or were receiving another induction therapy. The median  FC for all 14 patients after food reintroduction was 900 mg/kg . In Group A, median  FC levels were 1,181  mg/kg and in Group B 411  mg/kg  and as percentage of RNI   , their cumulative intake in Group A was significantly higher compared to Group B by a median difference of 87.7 g/day  . Nonetheless, the intake of gluten containing cereal products was higher in Group A than Group B, and in reverse, the consumption of non-gluten containing cereal products was higher in Group B . The food groups ‘alcoholic beverages’, ‘commercial toddlers’ foods and drinks’ and ‘nuts and seeds’ were not consumed by any of the patients and were therefore excluded from analysis.Although the intake of red meat and processed meat, when assessed separately, did not differ between the two groups (p = 0.047), thiamine (mg)  and niacin (%RNI)  had a moderate-strong positive correlation with FC , meat and meat products  and the intake of non-alcoholic beverages which included juices, and fizzy drinks  , Table p = 0.121, p-adj = 0.211), although most patients in Group A had GIP measurements above the upper detection limit of the assay, suggesting high gluten intake. There was no difference in faecal starch output between the two groups .Butyrate levels, expressed either as absolute concentration (µmol/g) or as relative proportion (%), were significantly higher in patients with higher FC levels (Group A), compared to patients with lower FC levels (Group B) by a median difference of 28.4 µmol/g and 6.4% respectively , but not with cereal products, nor vegetables indicates that protein from animal sources is likely to explain the negative relationship with FC we observed in the current study. High intake of animal protein has been associated with development of IBD [Patients in the current study with higher FC levels reported a higher protein and phosphorus intake, along with a higher intake of red and processed meat. Although we did not assess separately the intake of protein from animal or plant sources, the presence of a moderate-to-strong correlation between the intake of protein and meat products  [In the current study, double levels of faecal butyrate levels were observed in patients with higher FC levels after food reintroduction. This signal was associated with higher dietary fibre intake from which butyrate originates as an end-product of bacterial fermentation in the same group  . Higher = 0.051)  and a si= 0.051) , 43. TheAlthough patients in Group A reported higher protein intakes compared to those in Group B, there were no significant differences in BCFA levels between the two groups. This could potentially result from adequate intake of dietary fibre in those patients, which exceeds the threshold below which excessive protein fermentation occurs in the colon .The small sample size is the main limitation of this pilot study. This was due to the modest number of patients who met our stringent inclusion criteria and few patients who did not return dietary records paired with faecal samples. However, selection of a homogenous population with all patients enrolled at the end of an EEN course and while they were still in clinical remission, without receiving other concomitant induction treatment, minimised variance in our measured outcomes and increased statistical power. Beyond the dietary analysis presented here, the observed dietary signals of the current study might also represent biomarkers of unidentified covariates of other food ingredients which might be important in gut inflammation in CD. An indicative example could be baker’s yeast, which is present in bread and bakery products, constitutes a significant proportion of the cereal and cereal-based products group, and has been previously implicated in aggravating disease activity in CD . It is aThe current pilot study for first time in the literature identified that a diet higher in dietary fibre, protein, and red processed meat was associated with higher levels of intestinal inflammation after early food reintroduction in children with CD, post-EEN. These data need confirmation in larger, prospective studies to gain more insight into the dietary triggers of gut inflammation in CD, including the role subtypes of dietary fibre, and any mediating role the microbiome may have. Such research would help identify dietary components causing inflammation in CD and enable development of food reintroduction and personalised dietary regimes, leading potentially to improvements in CD management and prolonged clinical remission. A clinical trial like that is currently undergoing .Additional file 1: Fig. S1 (.png) Faecal calprotectin values before the start of EEN treatment and at 4 weeks and 8 weeks of EEN treatment in all 14 patients. Different colours indicate different patients.Additional file 2: Fig. S2. (.pdf) Hierarchical dendrogram showing the classification of individual food groups to larger level food groups, based on the National Diet and Nutrition SurveyAdditional file 3: Fig. S3. (.tif) Stratification of patients based on the median faecal calprotectin (900 mg/kg) of the entire group at food reintroduction. Group A (n=7): above median faecal calprotectin, Group B (n=7): below median faecal calprotectin.Additional file 4. Supplementary Tables 1, 2, 3.


Significant differences were found in reparative dentin formation between the treatment groups (p = 0.0005). Dentin bridge formation was observed in the MTA-treated group. The local action of HA/β-TCP/C is similar to that of MTA when used as an agent for pulp vital treatment in terms of absence of inflammation and maintenance of pulp vitality, although there are significant differences between both materials regarding the formation of dentin bridges.Bioceramic materials possess desirable biological properties, highlighting their non-reactivity and osteoconductivity. Their use has been extended in vital pulp treatment. The purpose of this study was to evaluate and compare the effects of beta-tricalcium phosphate (β-TCP), hydroxyapatite (HA), and collagen (C) scaffold with mineral trioxide aggregate (MTA) on the vital pulp of rat molars. Thirty-two molars of Sprague–Dawley rats underwent direct pulp capping with β-TCP/HA/C (n = 16) and MTA (n = 16). After 30 days, the following parameters were evaluated in the tested samples: the degree of pulp inflammation and pulp vitality, the presence of reparative dentin, the homogeneity of the odontoblastic layer, and the presence of pulp fibrosis. No statistically significant differences were observed between HA/β-TCP/C and MTA in terms of the degree of inflammation ( Hydroxyapatite (HA) and tricalcium phosphate (TCP) are categorized as bioceramic materials with excellent biological properties, among which their non-reactivity and osteoconductivity can be highlighted . Their uTCP is a porous bioceramic material which is resorbable and biocompatible. This material gradually degrades and becomes replaced by bone tissue. In this way, it acts as a scaffold for bone growth . Among tTCP and HA have been applied in several dental and maxillofacial procedures, including endodontic treatments ,8. The cCollagen (C) is a natural, bioabsorbable structural protein with which cells can interact and adhere. Collagen sponges and foams have been used as hemostatic agents, scaffolds for tissue repair, and for cell growth support . LikewisPrevious reports have confirmed that the incorporation of a 3D structure, β-TCP and collagen hybrid constructs favor the adhesion and proliferation of human dental pulp stem cells, thus promoting osteogenic/odontoblastic differentiation ,17. SimiFrom a biological perspective, it has been shown that some biomaterials like calcium silicate or β-TCPcan directly modify the osteoblastic proliferation rate and differentiation, such as the synthesis of alkaline phosphatase ,20,21. IAmong VPT procedures, direct pulp capping is indicated when the pulp is visibly exposed  due to caries, trauma, or iatrogenic stimuli such as accidental exposure during tooth preparation or caries removal . MineralA series of compounds with β-TCP have been tested in vitro on pulp cells to verify their biocompatibility and possible use as pulp capping materials . AdditioHA/β-TCP’s mechanism of action on osteoblasts in bone may be extrapolated to its action on teeth and odontoblasts. The purpose of the present study was to evaluate the response of vital pulp tissue in rat molars to HA/β-TCP/C when used as a capping agent and compare its histological effects to those of MTA. The null hypothesis is that there are no differences between HA/β-TCP/C and MTA in vivo.The Research Ethics Commission of the University of Murcia  approved this study (ID: 292/2017). Eight male Sprague–Dawley rats were used following Royal Decree 1201/2005, law 32/2007, European Directive 2010/63/UE on the protection of animals used for scientific purposes. The average weight of the rats was 230 g approximately, and their average age was 3 months. Rats were kept in cages with appropriate measures for adequate animal welfare and were fed with specific food for experimental animals  with an ideal composition, and with periodic toxicity and sanitary analyzes. The animals ate and drank ad libitum. Periodic health checks were carried out on the animals.®. Bayer. Kiel, Germany) and ketamine chlorhydrate 100 mg + clorobutanol 5 mg  were used to anesthetize the rats (dose = 0.2 mL/100 g weight). 0.12% chlorhexidine  was used to clean the molars’ surface. To access the cameral pulp, a tungsten carbide burr with a 0.8 mm-diameter pyriform shape was used , driven by a turbine with aqueous cooling . Hemostasis was echieved by pressure with sterile cotton balls for a maximum of 5 min. The experimental materials were placed on the pulpusing a small ball applicator. A small amount of test material was applied onto the exposure (approximately 400 ng), enough so that the exposure was sealed entirely by de material. Then, the excess moisture was removed with a dry cotton pellet. Subsequently, it was covered with a thin base of zinc oxide-eugenol  and the cavity was restored with silver amalgam  [Four pulp exposures were performed per rat in healthy first and second maxillary molars. Intramuscular injections with a mixture (at 50%) of 2% xylazine hydrochloride  .®  was used as a pulp capping material and was prepared by following its manufacturer’s instructions.-Group 1: MTA (n = 16 molars). Pro-Root MTA-Group 2: HA/β-TCP/C (n = 16 molars). The mixture was composed of 40% Hydroxyapatite , 30% β-TCP , and 30% collagen . All components were powder materials. A creamy consistency was achieved when mixed with distilled water. The particle size was 0.7 mm, and the pore size was 300 µm.To calculate the sample size (n = 16 per group), a sample size and power calculator was used . An α-ri® and Imalgene® 1000), and the fragments of maxillae containing the test molars were separated.The material was left to act over a period of 30 days. After this period, rats were sacrificed by a lethal injection of the two anesthetics mentioned above  to decalcify the hard tissues. Afterwards, specimens were dehydrated in graded alcohol, included in paraffin, and sectioned in 8 µm slices. Every five sections (40 μm) were stained with hematoxylin-eosin and Masson’s trichrome before optical microscope observation . 4 levels were analyzed per specimen.According to the histological evaluation criteria of Horsted et al. , later mp-values < 0.05 were considered significant.A descriptive statistical analysis of the histological data was carried out, finding the frequency distribution for each variable. Group data were compared by contingency table analysis using Pearson’s chi-square test and residual analysis to determine significant associations. In the MTA group, 16 teeth were evaluated. At thirty days, the histological analysis revealed a pulp tissue with the presence of fibrosis (100%) and a tubular reparative dentin in contact with MTA (87.5%) a. A reguIn the experimental group treated with HA/β-TCP/C, 16 teeth were also evaluated. Thirty days after treatment, the most common pattern was of a pulp with the presence of blood vessels, without inflammation or necrosis, and with a regular odontoblastic layer in 56.25% of the samples a; being p = 0.124). MTA group exhibited superior results in terms of reparative dentin and dentin bridge formation, with statistically significant differences. (p = 0.0005). Lastly, significant differences were identified for the odontoblastic layer (p = 0.003) and fibrosis (p = 0.03). All samples in the MTA group showed an intact odontoblastic layer and fibrosis  . The sequence of reparative processes resulting from the application of the tested pulp capping materials after the pulp chamber’s perforation were observed. In brief, the following sequence of pulp repair processes occur: In summary, according to the literature, during the first 15 days, a physiological inflammatory process occurs, which disappears in the following days to give rise to fibrosis . In the  = 0.03) b. These 0%. The sMTA releases calcium ions when it comes into contact with pulp cells, leading to the expression of osteonectin, bone morphogenetic protein, and osteopontin. MTA solubilizes growth factors such as TGF-b in order to form new tissues , and vasRegarding β-TCP, evidence describes that it can increase the adhesion and proliferation of human dental pulp stem cells and promote their osteogenic differentiation when used as a 3D-printed biomaterial . AtalayiIn fact, other authors affirm that β-TCP promotes dentin formation after pulp exposure in pig teeth. This process may be related to the release of calcium ions after the degradation of β-TCP in contact with pulp cells . HoweverThe effects of scaffold porosities on cell viability and differentiation of human dental pulp cells for dentin tissue regeneration were studied by Qader et al. . These aRegarding the results of this study, a healthy and structured pulp was observed with a regular odontoblastic layer both in molars treated with MTA (Group 1, 100%; This study’s main limitation was the lack of evidence regarding HA/β-TCP/C-based scaffolds and their potential for pulp-capping treatment. Further investigations on histological and cellular components are recommended to assess the HA/β-TCP/C-based scaffolds. A longer follow-up period is also needed to evaluate the dentin reparation/neoformation.HA/β-TCP/C could be potentially used as an agent for vital pulp therapy. The local action of HA/β-TCP/C is similar to that of MTA when used as pulp capping agent, in terms of the absence of inflammation and maintenance of pulp vitality. However, there are significant differences between both materials in terms of dentin bridge formation, favoring MTA. Further clinical studies are needed to confirm the results shown by animal studies.


The management of adenomyosis is challenging and limiting. Qiu's Neiyi recipe (Qiu) is a traditional Chinese medicine (TCM) prescription clinically used for endometriosis treatment in China, but the effect and mechanism of Qiu on adenomyosis are undefined.  An experimental adenomyosis model was induced in female neonatal ICR mice administrated with tamoxifen. The adenomyosis mice were divided into five groups: high-, middle-, and low-Qiu's group, danazol group, and model group. The mice just administrated with the solvent only (no tamoxifen or drugs) were served as the control group. After 28 days of administration, the body, uterine, spleen, and thymus weights of all mice were examined. Then, the myometrial infiltration and the expression of inflammatory factors were detected by histology examination, ELISA, and qRT-PCR in the uterus. In addition, the MAPK/ERK signaling pathway-related protein expression in adenomyosis mice was detected by immunohistochemical (IHC) staining, qRT-PCR, and western blotting. β, IL-6, and TNF-α in serum and uterus tissues of adenomyosis model mice were also decreased after Qiu treatment. The improvement of Qiu on the adenomyosis was achieved by inhibiting the activated MAPK/ERK signaling pathway, including reducing the mRNA and protein expressions of p-ERK/ERK, p-JNK/JNK, and p-p38/p38 in the uterus tissues.  In experimental adenomyosis mice, Qiu treatment improved the symptoms of adenomyosis by reducing the myometrial infiltration and increasing the index of spleen and thymus. The elevated levels of IL-1 Qiu alleviated the inflammatory reaction and uterus histological changes in mice with adenomyosis, and the potential mechanism is through the inhibition of the MAPK/ERK signaling pathway. Qiu may be a promising treatment for adenomyosis. Adenomyosis is a common disease occurring in reproductive-aged women. It can cause dysmenorrhea, pelvic pain, and abnormal uterine bleeding, which affects women's health and quality of life ,2. AdenoA previous study showed that adenomyosis is a kind of chronic inflammatory disease . VariousIn this study, a model of adenomyosis was established in ICR mice, then the effect and underlying mechanism of Qiu was evaluated on the development of adenomyosis. It was shown that Qiu could reduce the myometrial infiltration of endometrial implants and alleviate the inflammatory response in mice with adenomyosis by suppressing the activated MAPK signaling pathway. The current study may be meaningful for the application of Qiu as a clinical treatment for adenomyosis.Sargentodoxa cuneata (Oliv.) Rehd. et Wils. (Chinese pinyin name: daxueteng), 20 g LonicerajaponicaThunb (Chinese pinyin name: rendongteng), 20 g Spatholobus suberectus Dunn (Chinese pinyin name: jixueteng), 15 g Curculigo orchioides Gaertn. (Chinese pinyin name: xianmao), 15 g Epimedium brevicornu Maxim. (Chinese pinyin name: xianlingpi), 12 g Cistanche salsa (C. A. Mey.) G. Beck (Chinese pinyin name: congrong), 9 g Morinda officinalis How. (Chinese pinyin name: bajitian), 15 g Taraxacum mongolicum Hand.-Mazz. (Chinese pinyin name: pugongying), 15 g Salvia miltiorrhiza Bge. (Chinese pinyin name: danshen), 10 g Eupolyphagasinensis Walker (Chinese pinyin name: dibiechong), and 6 g Whitmania pigra Whitman (Chinese pinyin name: shuizhi). All herbs were identified by the pharmacists and provided by the department of pharmaceutical preparation of the First Affiliated Hospital of Zhejiang Chinese Medical University. All the herbs were weighed, mixed, and decocted with 10 folds of water at 100°C to obtain the final concentrations of 0.5 g/mL, 1 g/mL, and 2 g/mL, respectively.Qiu is composed of the following TCM herbs: 20 g n = 60) and their birth mother mice (n = 20) were purchased from the Centre of Experimental Animals at the Shanghai SLAC Laboratory Animal Co., Ltd.  with the approval number Certificate No. SCXK (Hu) 2017–0005. All the mice were housed in a standard room temperature of 23 ± 2°C and a humidity of 55–70% under a 12 h light/dark cycle with access to food and water. All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal experiments were performed according to the guidelines of laboratory animal care and were authorized by the Ethics Committee of Zhejiang Traditional Chinese Medicine University (Certificate No. SYXK (Zhe) 2018–0012; Hangzhou, China).Newborn ICR mice  were orally administered with 2.7 μmol/kg body weight of tamoxifen suspended in peanut oil/lecithin/condensed milk mixture  at a dose volume of 5 μL/g body weight from day 2 to day 5 after birth according to the previous study [n = 8) were fed with the equivalent amount of peanut oil/lecithin/condensed milk mixture without tamoxifen. The mice were weaned and separated from their mothers when they were 22 days old. Then, the mice were allowed to eat and drink freely until 3 months. After that, five model mice were randomly selected for pathological examination of uterine tissue, which confirmed the success of adenomyosis modeling.Female neonatal mice : the model group , the danazol group , the low dose of the Qiu group , the middle dose of the Qiu group , and the high dose of the Qiu group . At the same time, 8 aforementioned control neonatal mice were treated with solvent only and set as the normal group . Each group received relative treatment by daily intragastric administration for 28 days.After the treatment, the body weight of each group of mice was recorded. Then, these mice were sacrificed and their uterine, spleen, and thymus were excised. The weight of the uterine, spleen, and thymus were recorded. At the same time, the depth of myometrium invasion was also recorded and scored. The uteruses were postfixed overnight at room temperature in the same fixation solution, then embedded in paraffin for further analysis. The spleen index and thymus index were calculated as follows: spleen index (mg/g) = spleen weight (mg)/body weight (g), thymus index (mg/g) = thymus weight (mg)/body weight (g).β, IL-6, and TNF-α in the serum were detected by ELISA according to the manufacturer's instructions in the kit.After weighing the mice, blood was collected from the mice's eyeball and centrifuged at 3,000 rpm for 10 min, following which the supernatant was collected. The levels of IL-1μm and stained with HE. HE staining was scored to assess gland hyperplasia based on the following scale [Pathological damage of uterine tissue in each group of mice was detected by hematoxylin and eosin (HE). The uterine tissues were postfixed and embedded in paraffin, then sectioned at a thickness of about 4 ng scale : 0 point2O2. Then, the slides were incubated with primary antibodies overnight at 4°C. After washing, biotinylated secondary antibodies were added and incubated at room temperature for 20 min. Finally, the sections were stained with DAB to develop the color and counterstained with hematoxylin.An immunohistochemistry stain was performed to detect the expression of p-ERK, p-JNK, and p-p38 in uterine tissue. In brief, after being deparaffinized and rehydrated, the paraffin-embedded slides were extracted in a 0.1 mol/L citric acid buffer (PH = 6.0) and treated with 3% Hβ-actin by the 2−ΔΔCt method. The primer sequences used are shown in Total RNA was extracted from uterine tissue (myometrium and endometrium) using Trizol reagent  according to the manufacturer's instructions. Total RNA was reverse transcribed to cDNA and amplified and analyzed using SYBR Green PCR Master Mix  and a CFX96 real-time system . Each group was analyzed in triplicate. The original threshold cycle (Ct) values were standardized with After treatment, the uterine tissues (myometrium and endometrium) were lysed with RIPA buffer. After isolation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After blocking by 5% defatted milk powder, the membranes were incubated with the primary antibodies against tubulin alpha antibody , ERK1/2 antibody , phospho-ERK1/2 (Thr202/Tyr204) antibody , JNK1/2/3 antibody , phospho-ERK1/2 (Tyr204) antibody , p38 MAPK antibody , and phospho-p38 MAPK (Thr180/Tyr182) antibody  at 4°C for 24 h. After washing with PBS, the membranes were incubated with the secondary antibodies at room temperature for another 2 h. Lastly, the signals of protein bands were developed with enhanced chemiluminescence (ECL).P < 0.05 was considered as statistical significance.The statistical analyses were performed using SPSS 16.0 . Values are expressed as mean + SEM. Group comparisons were processed by using the one-way ANOVA analysis followed by Dunnett's post hoc test according to variance homogeneity and inconsistency. In all cases, P < 0.01). After being treated with M-/H-Qiu and danazol, the uterine weights significantly decreased (P < 0.05). Meanwhile, the mice given a high dose of Qiu treatment had lower uterine weight than the mice given low and middle doses of Qiu treatment. In addition, for the model group, the spleen index and thymus index significantly decreased, with the increased score of penetration of the ectopic endometrium into the midmyometrium compared to the control group (P < 0.01). Compared with the model group, after drug treatment, the spleen indexes were significantly increased in the high dose of Qiu and danazol, and the thymus indexes were significantly increased in the middle and high dose of Qiu and danazol (P < 0.05). However, the penetration of the ectopic endometrium into myometrium had no obvious improvement.The average food intake for all group mice had no differences. As shown in β, IL-6, and TNF-α) in serum and uterine tissue were measured by ELISA and qRT-PCR. As shown in Figures P < 0.05). The Qiu treatment inhibited the expression of IL-1β, IL-6, and TNF-α in the tamoxifen-induced adenomyosis mice in a dose-dependent manner. At the same time, the level of these proinflammatory mediators was also decreased after being treated with danazol (P < 0.01). In addition, the mRNA expressions of inflammatory factors  were significantly increased in the model group compared with those of the control group in the uterine tissue (P < 0.01). However, danazol and Qiu both suppressed the expression of these inflammatory factors (P < 0.01) . The hyperplasia of endometrial glands decreased, the glandular or interstitial components invaded the superficial myometrium of the uterus, and the structural changes improved significantly compared with the model group (The inflammatory infiltration and penetration of the ectopic endometrium were examined by hematoxylin-eosin (HE) staining. Compared with the control group, there was obviously endometrial glandular hyperplasia and adenomyosis in the myometrium in the model group. The glandular or interstitial components invaded the deep myometrium of the uterus. However, danazol and middle and high doses of Qiu dramatically alleviated this injury (el group .P < 0.01). However, the expression of these proteins was significantly decreased after being treated with danazol and Qiu (P < 0.05). And Qiu notably suppressed their elevated expressions in a dose-dependent manner.The expressions of p-ERK, p-JNK, and p-p38 in ectopic endometria were detected by immunohistochemistry. As shown in P < 0.01). However, the activation of the MAPK signaling pathway was inhibited by danazol and Qiu (P < 0.05). And Qiu notably suppressed the expression of these proteins in a dose-dependent manner. Nevertheless, there was no significant difference in the mRNA expression of MAPK signaling pathway-related genes in the endometrium  was significantly increased in the myometrium of model group mice (ometrium .P < 0.01). Danazol and middle and high doses of Qiu treatment decreased the expression of p-ERK, JNK, p-JNK, p38, and p-p38 significantly (P < 0.05). And the inhibition of Qiu on the activation of the MAPK signaling pathway was in a dose-dependent manner  can cause hyperalgesia and dysmenorrhea by inducing neuronal receptor activation. Although the studies about Qiu on adenomyosis-related inflammation are rare, the research studies on the herbs in Qiu also provides clues to its anti-inflammatory effect. Epimedium brevicornu Maxim. ethanol extract could exhibit an anti-inflammatory effect by inhibiting the production of several proinflammatory mediators, including NO, TNF-α, IL-1β, and IL-6 in LPS-induced peritonitis in vivo and in vitro [Morinda officinalis How. is reported to exert an anti-inflammatory effect in rheumatoid arthritis, which mainly contributes to the iridoids in it [β, IL-6 and TNF-α were increased in serum and uterine tissue of mice with adenomyosis compared to the controls, this funding was consist with the aforementioned results reported by others. More importantly, Qiu treatment remarkably abolished the inflammatory cytokine elevation in adenomyosis model mice, accompanied by an improvement of the adenomyosis symptoms. Based on the aforementioned studies of herbs or compounds in Qiu with anti-inflammatory effects, we indicated that our funding also confirmed the anti-inflammatory potential of Qiu in adenomyosis, which contributed to its therapeutic effect on adenomyosis.Inflammatory cytokines or cell infiltration is usually observed in the pathology of adenomyosis . IL-6 anin vitro . Morindads in it . In thisSargentodoxa cuneata, a herb of Qiu, has been reported to suppress the production of proinflammatory cytokines and the activation of the NF-κB, STAT3, and p38 MAPK in an acute lung injury model of mice and MH-S cells [β, IL-6, and TNF-α in serum and uterine tissue of adenomyosis mice. More importantly, the activated MAPK/ERK pathway could be suppressed by Qiu in a dose-dependent manner, the levels of inflammatory mediators as well as the disease severity were also decreased in our study.Meanwhile, the activation of proinflammatory mediators is critically associated with the regulation of inflammatory pathways in adenomyosis. The MAPK/ERK signaling pathway is proven to be activated in endometriosis and plays a key role in the inflammatory process and development of adenomyosis ,18. The In conclusion, our study showed that Qiu treatment reduced the myometrial infiltration, attenuated relative organ indexes, and decreased the levels of inflammatory factors in mice with adenomyosis. And this effect of Qiu on adenomyosis was associated with the inhibition of the MAPK/ERK pathway. However, the therapeutic effect of Qiu for adenomyosis remains not thoroughly studied. Therefore, further research for Qiu is required to comprehensively detect the potential benefits on adenomyosis.