Findings regarding the association between milk consumption and all-cause mortality reported by studies carried out in Western populations have been inconsistent. However, no studies have been conducted in Japan on this issue. The present study aimed to investigate the association of milk drinking with all-cause, cardiovascular, and cancer mortality in Japan.The data were obtained from the Japan Collaborative Cohort (JACC) study. A total of 94 980 Japanese adults aged 40–79 years who had no history of cancer, stroke, or chronic cardiovascular diseases were followed between 1988 and 2009. Multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) of mortalities were assessed using a Cox proportional hazard regression model and taking the lowest milk consumption group as the reference.During a median of 19 years of follow-up, there were 21 775 deaths . Drinking milk 1–2 times a month was associated with lower all-cause mortality in men compared to those who never drank milk . In women, those who drank 3–4 times a week also had a lower mortality risk compared with those who never drank milk . Inverse associations between drinking milk and mortality from cardiovascular diseases and cancer were found only in men.Drinking milk at least 1–2 times a month was associated with lower all-cause mortality in men compared to never drinking milk. An inverse association was also found between drinking milk and mortality from both cardiovascular diseases and cancer. However, lower all-cause mortality in women was found only in those who drank milk 3–4 times/week. The relationship of drinking milk or consumption of the nutrients found in milk and health has been often reported. For example, casein has been shown to have potent antimutagenic properties,6 coronary heart disease,7 endometrial cancer,8 ovarian cancer,9 and prostate cancer.10,11 Likewise, studies examining the association between milk consumption and all-cause mortality have produced inconsistent results. While an inverse association has been observed in a few studies,12–14 other investigations reported no association.15–18 In the baseline year of the present cohort study (1990), most “milk and dairy product” consumption (92.1%) was in the form of whole milk. However, the average milk consumption among Japanese at that time was much lower than that in Western countries.19However, on the contrary, some cohort studies have reported positive associations of milk consumption with stroke,Therefore, we aimed to examine the association between milk consumption and mortality from all and major causes in a large-scale community-based cohort of Japanese men and women, incorporating a wide range of potential confounding variables.20–22 Subjects with a previous history of any cancer, stroke, or chronic cardiovascular diseases  at baseline were excluded . Subjects who did not answer the question regarding milk consumption were also excluded . After these exclusions, 94 980 subjects (39 639 men and 55 341 women) were enrolled in the present analysis. The study design and informed consent procedure were approved by the Ethics Review Committee of Nagoya University School of Medicine.The Japan Collaborative Cohort (JACC) Study for Evaluation of Cancer Risks, sponsored by the Ministry of Education, Sports, Science, and Technology of Japan, started between 1988 and 1990, enrolling subjects living in 45 areas throughout Japan. A total of 110 585 subjects (46 395 men and 64 190 women) 40–79 years of age completed self-administered questionnaires about their lifestyles and medical histories. Sampling methods and other details of the JACC study have been described elsewhere.23,24 The Life Span Study showed that concordance between causes of death in death certificates and those of autopsy were 0.87 to 0.91 for cancer and 0.44 to 0.60 for cardiovascular disease.23 The Hisayama Study reported that concordance was 0.76 for all-cause mortality, 0.92 for cancer mortality, and 0.68 for cardiovascular mortality.24 We used the underlying causes of death coded by the International Statistical Classification of Diseases and Related Health Problems-10th Revision (ICD-10)25 to identify mortality endpoints: I00–I99 for mortality from cardiovascular disease (CVD) and C00–C97 for mortality from cancer.In each community, investigators conducted a systematic review of death certificates through 2009 for most communities . The date and cause of death were confirmed with the permission of the Director-General of the Prime Minister’s Office. Individuals who moved away from the study areas were treated as censored cases because subsequent deaths could not be confirmed. The registration of death is required by the Family Registration Law in Japan and is strictly observed. A few validation studies to date reported the accuracy of causes of death in death certificates.26 Specifically, the Spearman rank correlation coefficient between milk-drinking frequency and weighed dietary record for 12 days was 0.65, P < 0.001. We compared means using one-way analysis of variance and proportions using the chi-squared test.Information about milk drinking frequency and other lifestyle behaviors was obtained using a self-administered questionnaire. Subjects were divided into five groups by their self-reported milk-drinking frequency: “never,” “1–2 times/month,” “1–2 times/week,” “3–4 times/week,” and “almost every day” during the preceding year. The reproducibility and validity of the dietary questionnaire have been reported elsewhere.Sex-specific age-adjusted all-cause, CVD, and cancer mortality rates were assessed according to the five categories of milk drinking frequency using the Poisson regression model and were expressed as the rate per 1000 person-years. For each subject, the person-years of follow-up were calculated from the date that the baseline questionnaire was completed until the time of death, moving out of the community, or the end of follow-up, whichever occurred first. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated separately by sex using the Cox proportional hazard model adjusted for 5-year age groups.5,27–30 Missing values were treated as an additional category in the calculations.In multivariate analyses, the models were adjusted for potential confounding variables, including smoking status , alcohol drinking , sleep duration , body mass index , education level (attended school up to age 18), physical activity (exercise more than 1 hour per week), participation in health checkup in the preceding year, green-leafy vegetable intake , history of hypertension, history of diabetes mellitus, and history of liver diseases. The number of categories of the covariates adjusted in the multivariate models was dichotomous (yes or no) if not specified. These variables were selected as covariates because they were known or suspected to confound the association.n = 12 944), cohort 2 included subjects enrolled through 2003 (n = 4345), and cohort 3 included subjects enrolled through 2008 or 2009 (n = 77 691). These cohorts were treated as strata in the stratified Cox regression model adjusted for the same covariates as the multivariate model. Heterogeneity between the cohorts was also tested.Linear trends in mortality risks were assessed by assigning constants of 0, 1.5/30, 1.5/7, 3.5/7, and 1 to the ascending corresponding milk drinking frequency groups, and then treating the categories as numeric variables. Additionally, to address concerns regarding heterogeneity among study areas with different follow-up periods, meta-analysis using individual participant data was conducted. Specifically, three subgroups were defined as different cohorts: cohort 1 included subjects enrolled through 1999 . Statistical analyses were conducted with R version 2.15.3 ,31 using the Epicalc32 and survival packages.33 A P value of less than 0.05 was considered statistically significant.Stratified analysis by sex and age (40–64 and 65–79 years) using the same models was performed because milk-drinking habits may significantly differ with advancing age. Interaction for age groups by milk drinking frequency was tested by adding the interaction term to the Cox models using a likelihood ratio test. In an attempt to avoid possible reverse causation, secondary analysis was also conducted using the same models and excluding deaths that occurred within 5 years of baseline  dropped out of the follow-up (5.8%), and 21 775 died (12 203 men and 9572 women). Of these deaths, 28.8% were from CVD (26.1% in men and 32.2% in women), and 35.3% were from cancer (38.5% in men and 31.2% in women). The five most common sites of cancer death were lung, stomach, liver, pancreas, and colon in men , and stomach, lung, pancreas, liver, and colon in women .P = 0.09), although HRs for each category were still statistically significant. In women, both the trend and HRs for each category became insignificant after adjustment, except for the “3–4 times/week” category. Total CVD mortality rates were lower in men who drank milk 1–2 times/week or more compared to subjects who never drank milk: multivariable-adjusted HRs for each group compared to subjects who never drank milk were 0.86  for the “1–2 times/week” group, 0.89  for the “3–4 times/week” group, and 0.89  for the “almost every day” group (P for trend = 0.06). Only women who drank milk “3–4 times/week” had a marginally significant lower risk of CVD mortality  compared to those who never drank milk.Age and multivariable-adjusted HRs of all-cause, CVD, and cancer mortality according to the five milk-drinking frequencies are shown in Table P for trend = 0.56). In women, milk drinking frequency was not associated with cancer mortality.Cancer mortality rates were lower in men who drank milk 1–2 times/month or more compared to those who never drank milk: multivariable-adjusted HRs for each category were 0.88  for the “1–2 times/month” group, 0.90  for the “1–2 times/week” group, 0.85  for the “3–4 times/week” group, and 0.94  for the “almost every day” group. However, we did not observe any linear trend between milk drinking frequency and cancer mortality in men (P = 0.054) and significant in women (P = 0.022). All-cause mortality was inversely associated with the frequency of milk drinking in older men. Linearity of the association between milk drinking frequency and CVD mortality rates was not significant in either younger or older men, although the multivariable-adjusted HRs of younger men who drank milk “1–2 times/week” and “almost every day” compared to those who never drank milk were statistically significant: 0.81  and 0.88 , respectively. Similarly, although the HRs for cancer mortality were <1 in older men who drank milk 1–2 times/month or more compared to those who never drank milk, the association was not linear.Table Although we found lower age-adjusted all-cause and CVD mortality rates in women who drank milk 3–4 times/week compared to those who never drank in younger as well as older subgroups, HRs of milk drinking frequency with all-cause, CVD, and cancer mortality were not statistically significant after multivariate adjustment in any category in either age-group, including the “3–4 times/week” group. Results of the secondary analysis that excluded patients who died within 5 years of baseline were essentially the same  and 2.P = 0.09), a dose-response relationship was not evident between milk drinking frequency of 1–2 times/month or more and all-cause mortality. The associations were similar between milk drinking and both CVD and cancer mortality in men. In women, the risk of all-cause mortality was lower in those with a milk drinking frequency of 3–4 times/week than those who never drank milk.The present large prospective cohort study showed that men who drank milk at least 1–2 times/month had a significantly decreased risk of all-cause mortality compared to those who never drank milk. Although there was a suggestion of an inverse linear trend , could function to protect tissues from oxidative injury.On the other hand, inverse associations of milk drinking frequency with all-cause mortality were more evident in the older age group in both sexes  inhibition is one of the mechanisms that has been studied most in relation to the antihypertensive effect from milk-derived tripeptides.46 Results from in vitro experiments suggest that tripeptides derived from milk, such as isoleucine-proline-proline and valine-proline-proline,47 can inhibit ACE activity and potentially lower blood pressure. Furthermore, whey protein is rich in cysteine/cysteine and γ-glutamylcysteine dipeptides, which are efficient substrates for glutathione synthesis. Glutathione is a cellular antioxidant that destroys reactive oxygen species and detoxifies carcinogens.48 Milk fat also contains the highest level of naturally available conjugated linoleic acid among dietary sources, which could up-regulate the tumor suppressor gene in human breast cells49 and/or inhibit the expression of certain oncogenes.50–52 Other components in the milk, such as antioxidant vitamins , may bind and/or solubilize potentially oxidizing fatty acids or other agents.Our findings may be interpreted in two ways. First, a milk-drinking habit may simply indicate a generally healthy lifestyle. For example, milk-drinking frequency was negatively associated with the prevalence of smoking habit and positively associated with higher education and the frequency of vegetable intake in the present study. Although these variables were adjusted for in the multivariable models, there is a possibility of residual confounding. Second, nutritional components from milk itself may possibly explain the present results. For example, milk minerals, especially calcium, potassium, magnesium, and phosphorus, were inversely associated with hypertension.r = 0.65, P < 0.001).26 Nevertheless, further studies with more accurate assessments of milk in terms of types and portion size are warranted. Third, end of follow-up differed by area  in the current study, which might have introduced some biases. However, additional meta-analysis using individual data did not indicate heterogeneity (P = 0.55 for men and 0.61 for women); HRs were similar to the original model (data not shown).There are several limitations in the present study. First, information on milk drinking frequency and other lifestyles was self-reported and collected only at baseline. Second, types of milk , as well as the portion size per occasion, were not available from the FFQ used in the JACC study. However, milk drinking frequency was correlated with milk intake from 12-day weighed dietary records (In conclusion, the present large-scale prospective study found a significantly decreased risk of all-cause mortality in Japanese men who drank milk at least 1–2 times/month compared to never drinking milk. In contrast, among women, drinking milk 3–4 times/week, not every day, was associated with lower all-cause mortality compared to never drinking milk.eTable 1. eTable 2. Abstract in Japanese.


Polybrominated diphenyl ethers (PBDEs) are flame-retardant chemicals that are added to many consumer products. Multiple animal studies have shown PBDEs to be thyroid hormone (TH) disruptors. Epidemiologic evidence of PBDE exposure associated with TH disruption has been inconclusive.We used repeated measures to estimate associations between serum PBDE concentrations and THs in a North American adult cohort.From 2010 to 2011, we collected ≤ 3 serum samples at approximately 6-month intervals from 52 healthy adult office workers from Boston, Massachusetts, for analysis of PBDE congeners and THs.The geometric mean sum concentrations of the most prevalent PBDE congeners  were 22 ng/g lipid in winter 2010, 23 ng/g lipid in summer 2010, and 19 ng/g lipid in winter 2011. BDE-47 was the predominant congener. Based on a multivariable mixed regression model, we estimated that on average, a 1-ng/g serum increase in BDE-47 was associated with a 2.6-μg/dL decrease in total thyroxine (T4) . Total T4 was inversely associated with each PBDE congener. Serum concentrations of PBDEs were not strongly associated with total triiodothyronine (T3), free T4, or thyroid-stimulating hormone (TSH).These results are consistent with those from animal studies showing that exposure to PBDEs is associated with a decrease in serum T4. Because the other TH concentrations did not appear to be associated with BDE exposures, our findings do not indicate effects on the pituitary–thyroid axis. Taken together, our findings suggest that PBDE exposure might decrease the binding of T4 to serum T4 binding proteins.Makey CM, McClean MD, Braverman LE, Pearce EN, He XM, Sjödin A, Weinberg JM, Webster TF. 2016. Polybrominated diphenyl ether exposure and thyroid function tests in North American adults. Environ Health Perspect 124:420–425; http://dx.doi.org/10.1289/ehp.1509755 Polybrominated diphenyl ethers (PBDEs) have been used since the 1970s as additive flame retardants. The technical formulation pentabromodiphenyl ether (pentaBDE), composed of PBDE congeners containing three to six bromines, was added primarily to products containing polyurethane foam. Owing to their persistence in the environment, ability to bioaccumulate, and potential adverse health effects, pentaBDEs were added to the list of persistent organic pollutants in the Stockholm Convention in 2004 . U.S. ma4) and triiodothyronine (T3) (4 (TT4) concentrations  levels . PentaBDine (T3) . Multipltrations .in utero and during infancy, as well as for maintenance of many organ systems and metabolism throughout life  free thyroxine (fT4), total T3 (TT3), and thyroid-stimulating hormone (TSH). We also evaluated confounding and effect modification of these associations in our cohort.In the present study, we used repeated serum measures to assess the association between PBDE exposure and thyroid function in a longitudinal cohort of healthy adults. Our primary aim was to use these data to examine the association between PBDEs and thyroid function tests (TFTs) by measuring total TStudy design and population. We recruited 26 male and 26 female adult office workers living in the Boston (Massachusetts) metropolitan area and collected serum samples at approximately 6-month intervals from January 2010 to May 2011. This study is part of an extensive study of the effects of exposure to flame retardants on the health of office workers. Eligible subjects were required to be nonsmoking adults over the age of 18, self-described as generally healthy, and planning to reside in the Boston Metropolitan area for the duration of the study. Participants were excluded for having a prior diagnosis of thyroid disease or if they were pregnant. Characteristics and descriptions of the Flame Retardant Exposure (FlaRE) study population have been presented elsewhere  using established methods , TT3 , and TT4  . Thyroid peroxidase (TPO) antibodies were measured using an immunometric enzyme immunoassay (Orgentec Diagnostika) with the following reference ranges: normal < 50 IU/mL; borderline 50–75 IU/mL, and elevated > 75 IU/mL.Serum TFTs were measured at the Boston University School of Medicine. Enzyme-linked immunosorbent assays (ELISAs) were used to measure serum TSH , fTUrine samples. We collected 90 mL of urine from the participants at each sampling round. Urinary specific gravity was measured using a refractometer. We measured levels of urinary iodide, urinary perchlorate, and urinary thiocyanate using ion chromatography–mass spectrometry (IC-MS) with established methods . We used Spearman’s correlation coefficient to assess associations between PBDE congeners. All statistical analyses were performed using SAS statistical software , and statistical significance is reported at the 0.05 level.We used a general linear model with a random intercept to assess the association between PBDEs and TFTs, which in its simplest form isij =Y βo + β1PBDEij + bi + εij, [1]ijY represents the thyroid hormone level of the ith participant at the jth sampling round, βo is the fixed-effect intercept, β1 is an estimate of the mean difference in the TFT (or ln-TFT) with a 1-unit change in PBDE concentration, ib is the random intercept of the ith individual, and εij is the random error. The following covariates were added to Equation 1: time (indicator variables for sampling round), total lipids (milligrams per deciliter), urinary iodine concentration (UIC) (micrograms per liter), age (years), sex , body mass index , TPO antibody (yes/no), oral contraceptives (yes/no), perchlorate (micrograms per liter), thiocyanate (micrograms per liter), and specific gravity (SG). We selected potential confounding variables based on the a priori expectation that they could be associated with the exposure and the outcome but not be on the causal pathway. We assessed confounding using a change of 10% or more in the PBDE beta coefficient as a guide. We ran regression models on data sets excluding women taking oral contraceptives (n = 7) and participants who tested positive for the TPO antibody (n = 3) to assess their potential to affect regression estimates. We assessed effect measure modification by stratification on the following variables: sex and UIC (< 100 μg/L vs. ≥ 100 μg/L). For continuous covariates, we assessed possible effect modification by including a cross product of PBDE level and covariate .where We conducted regression analyses between PBDEs and TFTs with  and without  a participant who had experienced very high PBDE exposure and was identified as influential in this analysis with a Cook’s distance of 13.We estimated intraclass correlation coefficients (ICCs) to assess the stability of TFTs and UIC using an intercept-only general linear model with a random intercept. The ICCs were estimated by dividing the between-subject variance by the total variance.2 (r ≥ 0.85 for concentrations measured at the first visit), whereas BDE-153 was moderately correlated with the lower brominated PBDE congeners (r = 0.29–0.47) .The final study population for the thyroid analysis consisted of 26 males and 25 females. Participation rate by sampling round was 96% in round 1 (winter 2010), 96% in round 2 (summer 2011), and 81% in round 3 (winter 2011). The median age was 37 years old; 98% of the participants had a college degree; and 63% of the participants had BMI < 25 kg/m2 . Forty-f2 . GM conc4, TSH, fT4, TT3 and TPO. TFTs were predominantly within normal ranges (data not shown). Three female participants  had elevated TPO antibodies, which are markers for thyroid autoimmunity, but their TFT values were within normal ranges (data not shown). A sensitivity analysis that excluded these three participants did not alter the results (data not shown). The median UICs by sampling round were all > 100 μg/L, indicative of iodine sufficiency ; the remaining T4 (fT4) is unbound and available for deiodination in the outer phenolic ring to generate the bioactive hormone T3 (4 have mostly replaced measurements of TT4 (which is predominately bound hormone) as a measure of thyroid status in clinical practice  to adjust for serum lipids—lipids as a covariate, lipid-standardization—when studying the health effects of lipophilic chemicals . As expe4 in our iodine-sufficient study population.Iodide, an essential component of THs, has been reported to be an effect measure modifier in epidemiologic studies such that subgroups with insufficient iodine intake appear to be more vulnerable to xenobiotic effects on TH levels than iodine-sufficient subgroups . Iodide a priori criteria , and some samples were missing because participants completed only one (n = 2) or two (n = 9) of the three (n = 41) study visits, leaving a total of 137 serum samples from 51 participants. We believe that the likelihood that an individual sample was missing would have been unrelated to the actual TFT value of the missing sample, which would, on average, result in data that are missing completely at random (MCAR). If this assumption is correct for our population, missing data should not have biased associations because general linear models are robust to missing data that are MCAR  PDFClick here for additional data file.


The goal of our study is to evaluate the effect of Scutellarin on type II diabetes-induced testicular disorder and show the mechanism of Scutellarin's action. We used streptozotocin and high-fat diet to establish type II diabetic rat model. TUNEL and haematoxylin and eosin staining were used to evaluate the testicular apoptotic cells and morphologic changes. Immunohistochemical staining was used to measure the expression level of vascular endothelial growth factor and blood vessel density in testes. Oxidative stress in testes and epididymis was tested by fluorescence spectrophotometer and ELISA. The expression of Bcl-2/Bax and blood flow rate in testicular vessels were measured by western blot and Doppler. Our results for the first time showed that hyperglycemia induced apoptotic cells and morphologic impairments in testes of rats, while administration of Scutellarin can significantly inhibit these damages. This effect of Scutellarin is controlled by two apoptotic triggers: ROS/Bcl-2/Bax and ROS/microcirculation/starving pathway. Diabetes mellitus (DM) is one of the common metabolic diseases characterized by hyperglycemia. Global prevalence of DM was approximately 2.8% in 2000 and is estimated to reach 4.4% in 2030 . SustainIncreased oxidative stress in diabetes has been considered to contribute to the damage of reproductive system . OxidatiDM leads to vascular complications, associating with the appearance of macroangiopathy, microangiopathy, and diabetic thrombophilia in multiple organs . ActuallCurrently, insulin and oral hypoglycemic agents are commonly used for diabetes. Although big effort has been made for controlling the blood glucose, the complication of diabetes is still the major reason to cause organ dysfunction and death . Thus, n Erigeron breviscapus (Vant.) is one of the flavone glycosides. Hand.-Mazz. SCU 50 value of SCU could not be measured, and its maximum tolerance was 10 g/kg. Continuous oral intake of SCU (100 and 500 mg/kg) up to one month did not lead to any death or organ damage in rats [Recent studies show that many natural products have antidiabetic activity  and sign in rats . Because in rats . In thisn = 8), diabetes group (n = 8), and diabetes + SCU group (n = 8). Rats were fed under controlled environment at 25 ± 3°C, humidity of 40–65% and 12 h light/dark cycle, and free access to food and drink water. Normal food  was given to the control group. High-fat diet , containing 30% fat, 15% protein, and 55% carbohydrate, was used in diabetes group and diabetes + SCU group to induce insulin resistance. At week 8, a single dose of streptozotocin  dissolved in 0.1 M citrate-phosphate buffer was freshly prepared and intraperitoneally administered into diabetes group and diabetes + SCU group to induce the type II diabetes mellitus (HFD feeding was continued). The same volume of 0.1 M citrate-phosphate buffer was used as vehicle control for control group. The rats with blood glucose levels higher than 13 mmol/L for three times were considered as type II diabetes mellitus (T2DM). Blood obtained from rat tail-vein was evaluated to determine the levels of blood glucose using automated blood glucose analyzer . After one week of the injection of STZ, rats in diabetes + SCU group were intragastrically administrated with SCU  dissolved in a phosphate buffer  every day for 2 months. The dose of STZ and SCU was according to previous published work [All animal protocols were approved by China Council on Animal care and Sun Yet-San University Committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Adult male Wistar rats  were purchased from animal facility of Sun Yat-Sen University and housed in stainless steel cages. Rats were randomly divided into three groups: control group  at 37°C for 1 hour followed by rinsing the slides with phosphate-buffered saline to stop the reaction. Apoptotic cells were analyzed by randomly counting the TUNEL positive cells from sixty cross-sections of seminiferous tubule/slide under the fluorescence microscope at ×200 and ×400 magnification . Results were quantitative analysis as TUNEL positive cells per 100 cells (Image-Pro Plus 6.0).Apoptotic cells in testicular were measured through TUNEL assay with the In Situ Cell Death Detection Kit, POD  following the manufacturer's instructions. Testicular tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned at 5 Five sections from each group were cut consecutively from paraffin-embedded block. After dewaxing in xylene and dehydrating in graded concentrations of alcohol, sections were stained by haematoxylin and eosin (H&E) used to evaluate testicular morphology. The results were observed under light microscope at ×200 magnification.2O2 solution in methanol for 20 min. After three times washing with TBS, the slides reacted with anti-VEGF primary antibody  or anti-vWF primary antibody  and then reacted with anti-rabbit secondary antibody at 37°C for 30 mins followed by three washes with PBS, respectively. Diaminobenzidine  was used to visualize immunoreactive proteins. The protein levels were detected using Polink-2 Plus IHC Detection System  after staining with diaminobenzidine  to visualize immunoreactive proteins following the manufacture instruction. Diaminobenzidine (DAB) was added to the slides for 3 min as a chromogen. Followed by rinsing in running tap water, slides were counterstained with hematoxylin (blue). Finally, slides were dehydrated in absolute ethanol and mounted. All of them were observed under the light microscope at ×200 magnification and ×400 magnification .Immunohistochemistry was performed according to Sisman et al.  and the μL of DCFH-DA  was dropped into each sample. During 15 min incubation at room temperature, DCFH-DA can incorporate into any membrane-bound vesicles and esterases can cleave the diacetate group. At that time, 40 μL of the Fe2+ was put into the samples, which can transform the DCFH to fluorescent product DCF measured by fluorescence spectrophotometer  with excitation at 485 nm and emission at 530 nm.ROS were determined following the method of Driver et al. . Testes The concentration of MDA was considered as a marker of lipid peroxidation, which can reveal the oxidative damage as a result of diabetes. Testes were removed from rats under ethylether anaesthesia and were grinded by Tube Mill control . Homogenates were centrifuged at 4°C, 10000 rpm for 10 min. The supernatant was pipetted into 96-well plates and used for the ELISA test. The whole blood samples were taken from the abdominal vein of rats, and then they were spun to get serum. We detected the MAD level of testes and serum by Lipid Peroxidation (MDA) Assay Kit , following the manufacturer's instructions.After anaesthesia by chloral hydrate, the testicular vasculature was localized with a MS 400 probe (18–38 MHz) for color Doppler sonography  and Doppler waveforms were measured. The control steps were carried out following the manufacturer's instructions and previous published paper . The redμg) was added to each lane onto 10% SDS-polyacrylamide gels. After electrophoresis, we used Tris-buffered saline containing 0.1% Tween-20 to wash the polyvinylidene fluoride (PVDF) membranes  and then incubated them with primary antibody: anti-Bcl-2 , anti-Bax , anti-vWF , and anti-VEGF  at 4°C overnight. Secondary antibody  was added to the membrane for 2-hour incubation. Protein was visualized with Immobilon Western Chemiluminescent HRP Substrate . Band intensity was quantified by densitometry using the Image J software .Testis samples were immersed in lysed buffer and centrifuged at 13,000 rpm for 15 min at 4°C. Bicinchoninic Acid (BCA) Kit  was used to measure the protein concentration in the supernatants with steps according to the manufacturer's protocol. Total protein (50 Blood samples (5 mL) were taken from the inferior vena cava before sacrifice at the week 16. Serum was spun at 1500 rpm for 30 minutes and stored at −20°C until used. These sera were sent to clinical laboratory of the First Affiliated Hospital, Sun Yat-Sen University, analyzed for triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL), and low density lipoprotein (LDL).P < 0.05 was considered statistically significant.The data were shown as the mean ± standard deviation. Significant differences between the groups were determined using a one-way ANOVA through the SPSS 19.0 software. P > 0.05). The data of body weight and serum glucose from week 1 to week 16 were present as mean ± SEM .Body weights and serum glucose of experimental rats were showed in P ≤ 0.05). In the diabetes + SCU group, both of these weights were heavier than those of diabetes group (P ≤ 0.05).The testicular weights and epididymal weights of rats were presented in P < 0.05). The increased MDA and ROS can be effectively suppressed by SCU, while concentrations of MDA and ROS have significant difference between diabetes group and diabetes + SCU group (P < 0.05).MDA and ROS concentrations in testes, epididymis, and serum were shown in P < 0.05; An obvious increasing of testicular apoptosis in diabetic rats at 2 months after diabetes onset, measured by TUNEL staining Figures , was fouControl group showed the normal testicular structure and regular seminiferous tubular morphology with plenty germ cells and spermatozoa. The blood vessel walls in the control group were thin and smooth . In the P < 0.05). In the diabetes + SCU group, VTI was obviously increased to 131.25 mm/s (P < 0.05)  . Cytopla < 0.05) . SCU tre < 0.05)  Immunohi < 0.05) .DM, a state of chronic hyperglycemia, is a serious global disease with multiple organ and system dysfunction, including reproductive system . HyperglIn this study, we evaluated the effect of SCU, a traditional herbal medicine, which has been shown to diminish oxidative stress and improve microcirculation in some diabetic complications , on hypeSTZ combined with HFD is a common way to induce T2DM in rat. STZ induces inflation and degeneration of Langerhans islets beta cells, leading to hyperglycemia . Thus, h P ≤ 0.05), suggesting that downstream effector of hyperglycemia plays major role in the weight loss of reproductive organs in diabetic rat. In our study, ROS-induced apoptotic cell death and organ atrophy may be the major factor for the organ weight loss. It is noted that SCU totally reversed the weight loss of testes and epididymis but only partially blocked body weight loss (Body weight loss is a common symptom in STZ-induced diabetic rats, which is combined with organ weight loss. The reason for weight loss is that STZ possibly prevents the secretion of testosterone and growth hormone, resulting in disorder of anabolic activities . Data inght loss , suggestGrowing evidence indicates that oxidative stress is increased in diabetes due to overproduction of ROS and decreased efficiency of antioxidant defences . ROS, asROS-induced apoptosis is associated with ROS-induced mitochondrial DNA damage, leading to activation of DNA damage-mediated p53 signaling pathway, involving upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2 in testes of diabetic rats [Recent study showed that sulforaphane, a controller of detoxification response, targeted antioxidative factor, Nrf2, to inhibit diabetes-induced cell apoptosis pathway in testes . ActuallAlthough researches have proven that sulforaphane has effect on suppressing oxidative stress and cell death, but little evidence indicates that it can improve diabetes-induced microcirculatory disturbance of testes , 39. MicκB pathway, leading to vasospasm, enhanced coagulation of platelet and blood, and ischemic episodes [The mechanism underlying the blood flow abnormalities possibly relates to oxidative-induced endothelial dysfunction, leading to vasospasm and elevated blood viscosity . There iepisodes , 43. Thu Erigeron breviscapus, is the major effective ingredient of breviscapine, which is widely used in the treatment of cardiovascular diseases [SCU, a well-known polyphenolic flavonoid extracted from the Chinese traditional herb, Fleabane Compositae,diseases . It is adiseases , 45. SCUdiseases , blood cdiseases , and nepdiseases . SCU hasdiseases , 48. Duediseases , diabetidiseases , and diadiseases . HoweverPresent study, for the first time, demonstrated that SCU has the ability to block hyperglycemia-induced apoptosis, ROS, and microcirculation impairment in testes of STZ-induced diabetes rats. Since ROS are the major upstream event of microcirculation impairment and germ cells apoptosis in diabetes, in this study we could not clarify if SCU improves microcirculation via its antioxidant property, its ability to directly improve microcirculation, or both. In addition, we could not clarify if SCU blocks hyperglycemia-induced apoptosis via other mechanisms than ROS, such as its direct effect on improving microcirculation. Further study is needed to better understand the mechanisms of SCU's actions and try to answer the question: is SCU better than other antioxidants in improving diabetes associated reproductive disorder? Recent study showed that sulforaphane also can protect against T2DM-induced testicular apoptosis . In the Taken together, our data suggest that SCU possesses the potential to reverse diabetes associated male reproductive disorder, providing first in vivo evidence to support further clinic study to use SCU as a nontoxic drug to prevent and treat diabetes-mediated reproductive disorder. Data also suggest that targeting ROS is one of the mechanisms whereby SCU blocks hyperglycemia diabetes-induced apoptosis of germ cells and improves hyperglycemia-induced microcirculation impairment in testes. Further clinic study is needed to verify the SCU's action.Supplemental table 1A: Mean values of body weights of three experimental groups at every week. Data are presented as mean ± SEM. Weights of all groups are increased from week 1 to week 8. Weights of diabetes group and Diabetes + SCU group show a decreased tendency from week 9, while control group is keep on growing.Supplemental table 1B: Mean values of blood glucose of three experimental groups at 8-16week. Data are presented as mean ± SEM. Control group show a normal level of plasma glucose, but it in diabetes group and diabetes + SCU group are higher than diabetic level. Supplemental table 2: Comparison of serum TG, TC, LDL and HDL in three experimental groups. Data are presented as mean ± SEM. There are no differences between those groups.


Receiver operating characteristic (ROC) curve analysis showed that different S100B levels between schizophrenia and healthy participants can be used as a clinical diagnostic factor . Multiple linear regression analysis found that length of illness (Beta = −0.161), plasma levels of inflammatory regulation factors   were independently associated with concentrations of S100B . Therefore, our results suggest the possible function of S100B in pathogenesis of schizophrenia, and implicate the important role of autoimmune response and balance to glial dysfunction in patients with schizophrenia.Glial damage and immune dysfunction are involved in pathogenesis of schizophrenia. However, interaction between glial damage and immune dysfunction in schizophrenia is undefined. This study aims to compare plasma S100 calcium binding protein (S100B) levels between schizophrenia patients and healthy participants, and to determine if immune markers are independently related with concentration of S100B in schizophrenia patients. Forty-one schizophrenia patients and thirty-three healthy volunteers were enrolled. Enzyme-linked immunosorbent assay (ELISA) was used to assess the concentrations of plasma S100B and inflammatory markers. We found that concentrations of S100B were elevated in schizophrenia patients than healthy participants ( Schizophrenia is a severe mental illness with variety of symptoms that affects cognitive function, perceptual experiences, speaking and activities. Schizophrenia has become a severe public health problem and exerts enormous economic and personal costs worldwide.Despite the ongoing tireless research efforts, the etiology of schizophrenia is still not clearly understood. Multiple hypotheses such as neurodevelopment hypothesis46et al. found decreased S100B plasma levels in schizophrenic patients, but Rothermundt et al. reported increased S100B plasma levels in unmedicated and treated schizophrenic patients14During the past two decades, the associations between brain tissue damage, or glial cell dysfunction (astrocytes and oligodendrocytes) and schizophrenia have been repeatedly reported44101113141516et al. found that levels of S100B were positively correlated with negative symptomatology, but Schmitt et al. found that levels of S100B were negatively correlated with deficit symptoms15And, the correlations between S100B and schizophrenia subtypes, clinical characteristics were inconclusive too14151718Similarly, during these decades, numerous studies from different areas showed that immune dysfunction was related with central neural system, and involved in the pathogenesis of schizophreniaAmong the hypotheses of schizophrenia, it was reported that inflammation can alter neurotransmitterFurthermore, microglial cells were demonstrated to be major immunocompetent cells of the brain and play an important role in the regulation of neuronal proliferation and differentiation. Pro-inflammatory cytokines could activate the microglial cells and induce the production of S100B, which potentially could injure neurons. Whereas, anti-inflammatory cytokines are beneficial for repairing damaged neuronal tissuesWith these facts in mind, we hypothesized that inflammatory markers are independently associated with the concentrations of S100B in schizophrenia patients. This study intend to compare the plasma concentrations of S100B between patients with schizophrenia and healthy volunteers, and to explore if the levels of inflammatory markers , regulation factors  and complement factor 3, are associated with plasma levels of S100B in patients with schizophrenia.In this study, forty one patients hospitalized in Shanghai Mental Health Center during 2014 were recruited, who were diagnosed schizophrenia according to the International Classification of Diseases-tenth edition (ICD-10) diagnostic criteria. Diagnosis and interviews were carried out by a trained clinical psychiatrist by semi structured clinical interview and review of medical records. Inclusion criteria were: (i) Age between 18 to 65 years old; (ii) Positive and Negative Symptom Scale (PANSS) total scores ≥60; (iii) Patients were drug naïve or drug free for at least 4 weeks before enrollment; (iv) Ability to read the research contents. Exclusion criteria were: (i)Alcohol and/or substance dependence or ever diagnosed with other psychiatric disorders; (ii) Pregnant or lactating; (iii) Physical diseases ; (iv) Infectious, physical injury and autoimmune diseases, using anti-inflammatory drugs, corticosteroids or antibiotics in the recent four weeks; (v) Infectious and autoimmune diseases one week after enrollment; (vi) Lack of consensus on the diagnosis.Thirty-three healthy volunteers, with gender and age matched, were invited to participate into the control group, who were staff members and medical students in Shanghai Mental Health Center. All the healthy controls were volunteers and met the following criteria. Inclusion criteria were: (i) Age between 18 to 65 years old; (ii) Ability to read the research contents. Exclusion criteria were: Family history of psychiatric disease, and some of the exclusion criteria of patients: from ii to v.This study was approved by the Ethical Committee of Shanghai Mental Health Center, Shanghai, China. All the methods and procedures of this study were carried out in accordance with regulations and guidelines established by this committee. Informed and written consent were gained from all participants.The symptoms and severity of patients were assessed using PANSSBlood samples were drawn on admission at about 8 a.m. from all subjects who were included in our study by venipuncture into a vacuum tube with Ethylene Diamine Tetraacetic Acid (EDTA). Samples were collected and centrifuged at 3000 g rates for 15 min at 4 °C, and plasma was obtained and stored at −70 °C until completion of the samples. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the plasma levels of S100B and inflammatory markers. The values of intra- and inter-assay coefficients were around 5%. Concentration is expressed as pg/ml , μg/ml (C3) and ng/ml (TGF-β1).Data was analyzed using IBM Statistical Product and Service Solutions version 19.0 (IBM SPSS 19.0). Kolmogorov-Smirnov test was used for assessing normality of the variables distributions. Variables which were not normally distributed were log-transformed to be normally distributed. Data were presented as mean ± standard deviation (SD) and as median and inter-quartile range for normal distributed variables and non-normal distributed variables, respectively. The distribution of categorical variables was compared with two groups using chi-square tests.t test. Binary logistic regression was used to measure the effect of significant variables that had been tested by univariate analyses among the patients with schizophrenia. Diagnostic accuracy of the plasma S100B was assessed by Receiver Operating Characteristic (ROC) curve analysis. Pearson's correlation coefficient was used to evaluate possible relationships between logS100B and PANSS scores . Associations between predictor variables and level of plasma S100B were detected by multiple linear regression models. Only non-missing data were included in the modeling exercise. All statistical tests were two tailed and the criterion for significance level was set at p < 0.05.Difference of normally distributed variables between the two groups was compared by 41 patients and 33 controls were enrolled in our study. Demographic and clinical data at base line of the two groups are showed in t = −2.152, P = 0.035)  , Fig. 1.p = 0.015)  was 0.666  , when as= 0.015) .r = −0.383, p = 0.014), general score  and the lack of response of PANSS , level of IL-17 (p < 0.001), logIL-23(p < 0.001), logIL-10(p < 0.001) and plasma level of logS100B. However, after multivariate regression analysis, independently associations were found between length of illness , TGF-β1 , logIL-23, logIL-10  with plasma levels of logS100B .Characteristics including gender, current age in years, education, job, marital status, age of first onset, length of illness, number of episodes, and inflammatory markers including hsCRP, C3, TGF-β1, IL-17, IL-23, IL-10 were selected to be the independents for linear regression analysis. Values of IL-23 and IL-10 which were non-normally distributed were log-transformed to normalize their distributions. Significant positive correlations were found between education .


This study evaluates the association between Internal Addiction (IA) and psychiatric co-morbidity in the literature.Meta-analyses were conducted on cross-sectional, case–control and cohort studies which examined the relationship between IA and psychiatric co-morbidity. Selected studies were extracted from major online databases. The inclusion criteria are as follows: 1) studies conducted on human subjects; 2) IA and psychiatric co-morbidity were assessed by standardised questionnaires; and 3) availability of adequate information to calculate the effect size. Random-effects models were used to calculate the aggregate prevalence and the pooled odds ratios (OR).Eight studies comprising 1641 patients suffering from IA and 11210 controls were included. Our analyses demonstrated a significant and positive association between IA and alcohol abuse , attention deficit and hyperactivity , depression  and anxiety .IA is significantly associated with alcohol abuse, attention deficit and hyperactivity, depression and anxiety. Internet addiction (IA) was initially considered a new psychiatric disorder in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) . FollowiWith the growth of internet users worldwide, IA has become a pandemic in the new era . Ko et aOnline databases were searched from inception to June 2012: PubMed (from 1966), Embase (from 1980), PsychINFO (From 1806), BIOSIS (from 1926), Science Direct (from 2006) and Cochrane CENTRAL (from 1993). The search terms used were permutations of keywords for internet addiction  and psychiatric co-morbidity  where the symbol* indicates truncation. Abstracts presented in major international conferences and unpublished dissertations were manually searched, and the authors of selected correspondences were contacted for further information. References from the retrieved papers were hand-searched.We sought all cross-sectional, case-control and cohort studies that examined the relationship between IA and psychiatric co-morbidity. Studies were included if they 1) were human studies involving patients suffering from IA and healthy controls without IA; 2) adopted a formal definition of IA based on the Young’s Internet Addiction Test, Chen Internet Addiction Scale or other well-defined criteria. Young’s Internet Addiction Test defines IA predominantly by 1) withdrawal; 2) social problems; 3) time management and performance; and 4) reality substitute . Chen InStudies were excluded if they 1) did not provide sufficient information to calculate the aggregate prevalence and OR; 2) did not provide a specific definition of, and criteria for IA; and 3) the authors did not respond to provide further information upon request including the psychiatric co-morbidity directly related to internet use (e.g. online gambling). Articles with abstracts that were written in the English or Chinese language but had full texts written in non-English or non-Chinese languages were excluded , year of publication and journal name) prior to selection. Selection of relevant publications was conducted independently by two authors (i.e. TYT and MWZ). Articles were initially screened on the basis of titles and abstracts. The short-listed articles were then evaluated on study design, and screened for the inclusion and exclusion criteria listed above. Disagreement regarding the inclusion or exclusion of retrieved papers was resolved by discussion with the first author (i.e. RCH) before the list of articles to be used in the meta-analysis was formalised. There were 13 studies published in Chinese  publication details ; b) the number of IA patients and healthy controls; c) the number of IA cases and controls for each psychiatric comorbidity; d) source of healthy controls; e) descriptions of instruments for assessment of psychiatric co-morbidity; and f) demographics of participants including mean age, proportion of gender, and proportion of ethnicities.2 were reported for the IA group and the healthy controls. To test the hypothesis that a specific psychiatric comorbidity was in fact more common in the IA group, the pooled OR was calculated. The pooled OR, 95% CI, z-value and P-value were reported for each figure of psychiatric comorbidity. Significant statistical difference was set at P < 0.05 for all analyses. Between-study heterogeneity was assessed with the I2 statistic, which describes the percentage of variability among effect estimates beyond that expected by chance [2 values of 25% was considered low, 50% moderate, and 75% high. Egger’s regression tests were performed to detect publication bias. If significant publication bias was present, the classic fail-safe test would be performed to determine the number of missing studies required for the P-value of publication bias among the observed studies to approximate > 0.05. Forest plots  adolescents (10-18 years of age); 2) young adults (19-39 years of age); and 3) middle-aged adults and the elderly (40-76 years of age). The age ranges of the aforementioned groups were determined by the mean age and overall age range of the subjects recruited.Eight studies comprising a total of 1641 patients with IA and 11210 controls without IA were included in the analysis Figure . The stu2 = 0.57, I2 = 94.18). The prevalence of alcohol abuse among the normal controls was 4.3% . Figure 2 = 0.052; Q = 4.22, df = 2, P = 0.121, I2 = 52.6).The prevalence of alcohol abuse among IA patients was 13.3% . The prevalence of ADHD among the normal controls was 8.9% . Figure 2 = 0.029; Q = 4.68, df = 3, P = 0.197, I2 = 35.9).The prevalence of ADHD among IA patients was 21.7% . The prevalence of depression among the normal controls was 11.7% . Figure 2 = 0.062, Q = 9.1, df = 4, P = 0.06, I2 = 56.1).The prevalence of depression among IA patients was 26.3% . The prevalence of anxiety among the normal controls was 10.3% . Figure 2 = 0.293; Q = 16.0, df = 3, P = 0.001, I2 = 81.2).The prevalence of anxiety among IA patients was 23.3% , depression  and anxiety .There was no significant publication bias for any of the psychiatric co-morbidity: alcohol abuse , ADHD  , the seqThe present meta-analysis engendered a significant association between IA and alcohol abuse, ADHD, depression and anxiety. Psychiatric co-morbidity occurs in a range from 13.3% to 26.3% among patients suffering from IA. To enhance understanding of the relationship between IA and psychiatric co-morbidity, prospective studies involving other psychiatric co-morbidities are required to establish the cause-and-effect mechanisms between IA and psychiatric co-morbidity. In view that the complex interactions between IA and psychiatric co-morbidity may lead to significant health burden, concerted mental health services need to be developed in anticipation of the recognition of IA as a diagnosable psychiatric disorder in the future.The authors declared that they have no competing interests.RCH contributed to conception and design, analysis and interpretation of data, drafting the article and revising it critically for important intellectual content, and final approval of the version to be published. MWZ contributed to analysis and interpretation of data, and drafting the article. TYT contributed to drafting the article and interpretation of data. AHT, FP, YL, CC, PSY, LTL, CML, HW revised the article critically for important intellectual content. KKM contributed to conception and design, analysis and interpretation of data, revising the article critically for important intellectual content, and final approval of the version to be published. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/14/183/prepubSummarizes all studies published in Chinese language and the reasons for exclusion.Click here for fileSummarizes all studies published in Korean language and the reasons for exclusion.Click here for file


Rhazya stricta and Zingiber officinale, respectively, on the growth of human GBM cell line, U251. R. stricta and Z. officinale are traditionally used in folkloric medicine and have antioxidant, anticarcinogenic, and free radical scavenging properties. Combination of CAERS and CFEZO treatments synergistically suppressed proliferation and colony formation and effectively induced morphological and biochemical features of apoptosis in U251 cells. Apoptosis induction was mediated by release of mitochondrial cytochrome c, increased Bax : Bcl-2 ratio, enhanced activities of caspase-3 and -9, and PARP-1 cleavage. CAERS and CFEZO treatments decreased expression levels of nuclear NF-κBp65, survivin, XIAP, and cyclin D1 and increased expression level of p53, p21, and Noxa. These results suggest that combination of CAERS and CFEZO provides a useful foundation for studying and developing novel chemotherapeutic agents for the treatment of GBM.Hitherto, limited clinical impact has been achieved in the treatment of glioblastoma (GBMs). Although phytochemicals found in medicinal herbs can provide mankind with new therapeutic remedies, single agent intervention has failed to bring the expected outcome in clinical trials. Therefore, combinations of several agents at once are gaining increasing attractiveness. In the present study, we investigated the effects of crude alkaloid (CAERS) and flavonoid (CFEZO) extracts prepared from medicinal herbs,  Glioblastoma multiforme (GBM) is the most common primary central nervous system neoplasm, accounting for more than half of all brain tumors, and is among the most lethal phenotypes of all cancers. Despite significant advances in molecular analysis and several promising therapies for GBM, the prognosis for patients with GBM remains poor . The poo c from the mitochondria to the cytoplasm. Both extrinsic and intrinsic pathways eventually converge on a common pathway, or the execution phase of apoptosis, activation of caspase-3 that provokes engagement of the effector caspases [Apoptosis is a highly sophisticated and elaborate mode of cell death that requires precise regulation of different intracellular signaling pathways to ensure the continuation of the transmission of the death signal. Apoptosis is tightly regulated by two opposing groups of hub proteins: death antagonists  and death agonists (Bax and Bak and other proteins) . The typcaspases . These lcaspases .Recently, considerable attention has been focused on dietary and medicinal phytochemicals derived from natural sources, as a rich reservoir for discovery of novel anticancer drugs . NonetheRhazya stricta Decne , a member of the Apocynaceae family, is an important medicinal species used in folkloric medicine to cure various diseases in South Asia and the Middle East [ R. stricta leaves have been prescribed for the treatment of various disorders [ R. stricta is a good source of antioxidants [ R. stricta inhibited cell proliferation and induced apoptotic cell death in the breast cancer cell lines MCF-7 and MDA MB-231 [ R. stricta and their anticancer activities have been demonstrated [ R. stricta is an alkaloid-rich herb deserves attention for many reasons. First, alkaloids are among the most important active components in natural herbs, where several alkaloids, isolated from natural herbs, have been shown to exhibit antiproliferation and antimetastasis effects on various types of cancers both in vitro and in vivo [ Catharanthus roseus  [ R. stricta have been found to exhibit numerous biological activities such as antimicrobial and antihypertensive activities [ R. stricta inhibited cell growth and sensitizedhuman lung cancer cells, A549, to cisplatin through induction of apoptosis [ R. stricta induced the chemopreventative enzyme, Nqo1, which could be, at least in part, a novel mechanism for the traditional use of R. stricta's alkaloid as an antitumor agent [ Rhazya's alkaloids will be necessary for credible assessments of the cancer chemopreventive qualities of the herb.dle East , 12. Extisorders , 12. It oxidants . We prevA MB-231 . Althougnstrated , 12, newnstrated . The fac in vivo . Seconds in vivo  and vinc family) , have altivities  and antitivities , 18. Recpoptosis . Finallyor agent . TherefoZingiber officinale Rosc. (Ginger), a member of the Zingiberaceae family, has been used in traditional oriental medicine for centuries to treat various gastrointestinal illnesses, arthritis, rheumatism, pain, muscle discomfort, various cardiovascular diseases, and metabolic diseases [ Z. officinale and have paid little attention, if any, to flavonoids. Flavonoids display a wide range of pharmacological properties, such as antimicrobial, antiviral, anti-inflammatory, antiallergic, analgesic, antioxidant, and hepatoprotective activities [ in vitro and in vivo cancer chemopreventive and chemotherapeutic potentialities [c with subsequent activation of caspase-9 and caspase-3, to increase levels of caspase-8, to downregulate expression of Bcl-2 and Bcl-xL, to enhance expression of Bax and Bak, and to modulate activity of nuclear factor-κB . Des Z. officinale or R. stricta in other cancer cells but no reported evidence on GBM cells. In the present study we have evaluated the antiproliferative potentialities and mechanisms of crude flavonoids and alkaloids isolated from Z. officinale or R. stricta, respectively, and found to be highly effective in GBM cell line.To our knowledge, previous studies have shown the chemopreventive effect of R. stricta leaves was prepared essentially as described elsewhere [ R. stricta (350 g) were soaked in 80% methanol (1 L) at ambient temperature for seven days after which the methanolic extract was evaporated in a rotatory evaporator and the remaining residue was suspended in water and filtered. The aqueous extract was then acidified with 10% glacial acetic acid and extracted with chloroform. This chloroform fraction contained weakly basic alkaloids and neutral compounds. The remaining aqueous solution was alkalinized using NaOH and the pH was adjusted to 11. The alkaline aqueous layer was extracted with chloroform to yield a chloroform fraction enriched in strongly basic alkaloids. The chloroform layer was evaporated to dryness to obtain a crude extract of alkaloids. Before use, the stock was further diluted in DMSO to give the final indicated concentrations and termed as crude alkaloid extract of R. stricta (CAERS).A crude alkaloid extract oflsewhere . Briefly Z. officinale, a rhizome of the herb was purchased from local market and powdered. The dried powder was extracted by cold percolation with 70% (2 L) ethanol for 72 h at room temperature and then filtered. The extraction was repeated twice. The combined filtrates were concentrated in a vacuum evaporator to afford a syrupy brown residue. This residue was suspended in 250 mL hot water (60°C), for h, filtered, and defatted by using petroleum ether (250 mL × 3). The aqueous portion was then separated, collected, and fractionated with N-butanol saturated water (250 mL × 3). The aqueous portion was discarded and the N-butanol portion was then separated, collected before being fractionated with 1% KOH. The KOH portion was then fractionated with dilute HCl (2%) and N-butanol saturated water. The dilute HCl portion was discarded. The N-butanol portion was then separated, collected, and dried to obtain a crude extract of flavonoids. Before use, the stock was further diluted in DMSO to give the final indicated concentrations and termed as crude flavonoid extract of Z. officinale (CFEZO).For preparation of crude flavonoid extract fromPhytochemical examination for testing the presence of alkaloids in CAERS was carried out using Dragendorff's test and Mayer's test. Few quantity of each portion was stirred with 5 mL of 1% aqueous HCl on water bath and then filtered. Of the filtrate, 1 mL was taken individually into 2 test tubes. To the first portion, few drops of Dragendorff's reagent (solution of potassium bismuth iodide) were added; occurrence of orange-red precipitate was taken as positive. To the second 1 mL, Mayer's reagent (potassium mercuric iodide) was forming a buff-colored precipitate indicating the presence of alkaloids .Phytochemical examination for testing the presence of flavonoids in CFEZO was carried out using sodium hydroxide test. Few quantity of the each portion was dissolved in water and filtered; to this 2 mL of the 10% aqueous sodium hydroxide was later added to produce a yellow colouration. A change in colour from yellow to colourless on addition of dilute hydrochloric acid was an indication of the presence of flavonoids .The human GBM, cancer breast (MCF-7), cervical carcinoma (HeLa), and nonmalignant human foreskin fibroblast (HF-5) cell lines were obtained from King Fahd Center for Medical Research, King Abdulaziz University, Saudi Arabia. The cells were grown in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin antibiotics (Promega) in tissue culture flasks under a humidifying atmosphere containing 5% CO2 and 95% air at 37°C. The cells were subcultured at 3-4-day interval.4/well in 96 wells plate for 24 h; then cells were treated with the indicated concentrations of the extracts and incubated for 24, 48, or 72 h. After the specified time of treatment, 10 μL of freshly prepared WST-1 solution was added to each well. Culture medium and WST-1/solution were added in an empty well as a blank for the microtiter plate reader. The absorbance of the treated and untreated samples was measured after 2 hours by  micro plate reader, at 450 nm with a reference wavelength 630 nm to avoid the interference of cell layer absorbance that blocks light passing through. In all experiments, the effects of CAERS and/or CFEZO on growth inhibition were assessed as percent cell viability, where nontreated cells were taken as 100% viable. For these studies, all experiments were repeated three or more times in triplicate.Cell viability and the effects of CAERS and/or CFEZO on the growth of U251 cells were assessed by tetrazolium salt WST-1 kit  following the manufacturer's instructions. Briefly, U251 cells were seeded, at a density of 10For clonogenic assay, approximately 500 cells were seeded into six-well plates in triplicate and allowed to adhere overnight. Thereafter, cell culture media were changed and cells were treated with indicated concentrations of CAERS and/or CFEZO extract(s). The cells were allowed to incubate at 37°C in the incubator undisturbed for 15 days. During this period each individual surviving cell would proliferate and form colonies. On day 15, the colonies were washed with cold phosphate buffer saline and fixed with −20°C cold methanol before being stained with 0.1% Coomassie Brilliant Blue. The colonies that had >50 cells/colony were counted and expressed as percent control.For soft agar colony-forming assay, U251 cells were seeded at 5,000 cells per well in 0.35% top agarose (Promega) with a base agarose of 0.7% agarose supplemented with complete medium. Cultures were treated with indicated concentrations of CAERS and/or CFEZO extract(s) and incubated in a humidified incubator at 37°C for 3 weeks. Cells were then stained with 0.5 mL of 0.0005% crystal violet, and colonies were counted visually. All experiments were done in triplicate with two independent experiments.4) were plated on coverslips, allowed to attach overnight, and exposed to indicated concentrations of the CAERS and/or CFEZO extract(s) for 48 h. The cells were washed with PBS and fixed with 3.7% paraformaldehyde  in PBS for 10 min at room temperature. Fixed cells were washed with PBS and stained with 4,6-diamidino-2-phenylindole  solution for 10 min at room temperature. The cells were washed two more times with PBS and analyzed. The cells with condensed and fragmented DNA (apoptotic cells) were scored under a fluorescence microscope  using the magnification indicated in the figures.The nuclear morphological changes associated with apoptosis were analyzed using DAPI staining. Briefly, cells (2 × 104) were plated on coverslips, allowed to attach overnight, and exposed to indicated concentrations of the CAERS and/or CFEZO extract(s) for 48 h. After incubation, cells were trypsinized and centrifuged for 5 min at room temperature. Then, the supernatant was decanted and pellets were dried and fixed with 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Then, fixed samples were washed in 0.1 M sodium cacodylate buffer (pH 7.2) and postfixed in 1% osmium tetroxide in the same buffer. The cells were dehydrated in a graded ethanol series and propylene oxide. The resin infiltration was performed with a 1 : 1 mixture of propylene oxide and epon for 5 h, followed by 100% epon for another 5 h. Next, the material was embedded, followed by 48 h of polymerization. Thin sections were produced using an ultramicrotome (LEICA EM UC6) and these were stained with toluidine blue.Cells (2 × 104 cells/well) treated with the indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h, harvested, washed in PBS, centrifugated at 12,500 ×g for 5 min, and resuspended in binding buffer. Then, 5 μL of Annexin V-FITC conjugate and 10 μL of propidium iodide (PI) solutions were added to the cell suspension and the cells were incubated at room temperature for 10 minutes protected from light. The FITC/PI fluorescence intensity was measured by flow cytometry to differentiate between viable (annexin V-negative and PI-negative), early apoptotic , and late apoptotic (annexin V-positive and PI-positive) cells. The extent of apoptosis was quantified as percentage of annexin V-positive cells.The ability of CAERS and/or CFEZO treatments to induce apoptosis in U251 cells was determined using an Annexin V-FITC Apoptosis Detection Kit (Sigma), according to manufacturer's instructions. Briefly, U251 cells were seeded in 6-well plate (20 × 106 cells/100 mm dish) treated with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h were collected, washed in PBS, and centrifugated at 12,500 ×g for 5 min. Cell pellets were then lysed in 600 μL lysis buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100), kept on ice for 30 min, and centrifuged at 12,500 ×g for 20 min. The supernatant from the lysate was treated with 2 μL RNase A (20 mg/mL) at 37°C for 1 h, followed by proteinase K digestion, 2 μL proteinase K (20 mg/mL) at 56°C for 15 min and then phenol chloroform extraction and isopropanol precipitation. After centrifugation at 12,500 ×g for 20 min, the DNA pellets were dissolved in 20 μL TE buffer (10 mM Tris (pH 7.4) and 1 mM EDTA (pH 8.0)) and the concentration of DNA was determined spectroscopically. Then, DNA was resolved by electrophoresis on 1.5% agarose gel at 80 100 V, stained with ethidium bromide, and visualized by a UV transilluminator (BIO-RAD).DNA gel electrophoresis was used to determine the presence of internucleosomal DNA cleavage as described previously . Brieflyμg/mL EENS for 24 h in complete medium, and the comet assay was done as described earlier [3 cells in a volume of 75 μL of 0.5% (w/v) low-melting-point agarose were pipetted onto a frosted glass slide coated with a thin layer of 1.0% (w/v) agarose, covered with a coverslip, and allowed to set on ice for 10 min. Following removal of the coverslip, the slides were immersed in ice cold lysis buffer containing 2.5 mol/L NaCl, 10 mmol/L Tris, 100 mmol/L Na2-EDTA, and 1% (w/v) N-lauroylsarcosine, adjusted to pH 10.0, and 1.0% Triton X-100 was added immediately before use. After 2 h at 4°C, the slides were placed into a horizontal electrophoresis tank filled with buffer ) and subjected to electrophoresis for 30 min at 300 mA. Slides were transferred to neutralization buffer (0.4 mol/L Tris-HCl) for 3 to 5 min washes and stained with ethidium bromide for 5 min. After a final wash in double-distilled water, the gels were covered with glass coverslips. To prevent additional DNA damage from visible light, all the steps described above were conducted under a dimmed light. Slides were viewed and nuclei images were visualized and captured at 400x magnifications with an Axioplan 2 fluorescence microscope (Zeiss) equipped with a CCD camera (Optronics). Hundreds of cells were scored to calculate the overall percentage of comet tail-positive cells.CAERS and/or CFEZO-induced DNA damage was determined using the comet assay. Cells were treated with 25 and 50  earlier . Briefly3/well) onto 6-well plates and treated with indicated concentrations of CAERS and/or CFEZO extract(s) for 24 h. After this period, floating and adherent cells were collected (with care being taken that none of the floating cells were lost during washes) and pelleted by centrifugation . RNA extraction and reverse transcriptase-PCR were done as previously described [μg/mL) staining.Cells were seeded  onto 6-well plates, treated with the indicated concentrations of CAERS and/or CFEZO extract(s), and incubated for 24 h. After this incubation, the cells were collected by centrifugation, washed twice with cold PBS, and resuspended in 500 μL of ice-cold cytosol extraction buffer  containing a protease inhibitor cocktail . After 30 min incubation on ice, the cells were homogenized in the same buffer using a dounce homogenizer (30 strokes) and centrifuged . The supernatant was collected and centrifuged again  to collect the mitochondria-rich (pellet) and cytosolic (supernatant) fractions. The supernatant was used as cytosolic lysate while the pellet was suspended in lysis buffer  glycerol, and 1% Triton X-100) supplemented with a protease inhibitor cocktail  before being centrifuged to obtain the mitochondrial lysate. Proteins concentrations were determined with a BCA protein assay kit (Pierce) and equal amounts of protein fractions were subjected for further analyses as described below. For preparation of nuclear extracts, treated cells were collected by centrifugation and washed twice with cold PBS, and nuclear extract was prepared using Pierce BCA Protein Assay Kit (Thermo Scientific) following the manufacture's procedure.To detect cyteviously . Briefly3/well) onto 6-well plates, treated with the indicated concentrations of CAERS and/or CFEZO extract(s), and incubated for 24 h. The cells were washed three times with PBS and lysed in cold lysis buffer containing 0.05 mmol/L Tris-HCl, 0.15 mmol/L NaCl, 1 mol/L EGTA, 1 mol/L EDTA, 20 mmol/L NaF, 100 mmol/L Na3VO4, 0.5% NP40, 1% Triton X-100, and 1 mol/L phenylmethylsulfonyl fluoride (pH 7.4) with freshly added protease inhibitor cocktail . The lysates were collected and cleared by centrifugation, and the supernatants were aliquoted and stored at −80°C. The protein contents in the lysates were measured by BCA protein assay , as per the manufacturer's protocol. Western immunoblotting was done essentially as described elsewhere [β-mercaptoethanol, electrophoresed on 10% SDS-PAGE, and transferred to PVDF membranes. After transfer, the membranes were incubated with primary antibody against tested proteins, followed by incubation with a secondary horseradish peroxidase-conjugated antibody. Antibodies were purchased from Sigma. The membranes were developed by the enhanced chemiluminescence (ECL) detection kit (Amersham). In all experiments, the blots were stripped with stripping buffer  and reprobed with anti-β-actin (Spring Bioscience) antibody as a control for protein loading. Signals were detected with an enhanced chemiluminescence detection kit (Amersham).The western blot analyses were carried out as detailed previously . Brieflylsewhere . Brieflyhttp://www.ssric.org/trd/spss16). Results with a P value <0.05 were considered statistically significant. Univariate analyses with linear regression model were used to estimate the probabilities of suppression of malignant cells. Standardized coefficients (Beta) and their 95% CIs were computed for variables in the model.All experiments were done at least three times independently and in triplicate. The results were presented as mean ± standard deviation (SD) for continuous variables. Differences between samples were analyzed with by one-way analysis of variance (ANOVA), followed by post hoc test using SPSS16.0 software  or CFEZO  for 24, 48, and 72 h. The cytotoxicity assessed by WST-1 kit showed that CAERS or CFEZO efficiently inhibited cell viability in dose- and time-dependent manners  for CAERS were 200, 135, and 80 μg/mL after 24, 48, and 72 h, respectively, and for CFEZO were 150, 115, and 75 μg/mL after 24, 48, and 72 and 150 μg/mL, respectively. To find whether CAERS and CFEZO have synergistic antiproliferative potentialities, U251 cells were incubated with increasing doses of CAERS  and CFEZO  in combination. As shown in 50 value indicated higher synergistic effect of the combination of extracts; this because IC50 values after 24, 48, and 72 h of combined treatments were 40, 20, and 15 μg/mL. Although we found a lower IC50 value of combination of 40 μg/mL CAERS and 40 μg/mL CFEZO than combination of 20 μg/mL CAERS and 20 μg/mL CFEZO (μg/mL CAERS and 40 μg/mL CFEZO that showed too much cytotoxicity (necrotic cells) as we found in the in situ Wright staining (data not shown). Therefore, we selected 150 μg/mL CAERS and 100 μg/mL CFEZO for single treatment and 20 μg/mL CAERS and 20 μg/mL CFEZO for combined treatment in all subsequent experiments.Initially, we examined the sensitivity of U251 cells to different doses of both extracts. So, cells were treated with increasing concentrations of CAERS  in U251 cells. This assay measures the ability of tumor cells to grow and form foci in a manner unrestricted by growth contact inhibition as is characteristically found in normal, untransformed cells. As such, clonogenicity provides an indirect estimation of the tendency of tumor cells to undergo neoplastic transformation. To measure clonogenicity, U251 cells at a given cell density were plated onto multiple well tissue culture dishes, with and without addition of increasingdoses of CAERS or CFEZO. Control and treated cells were maintained in culture for an additional 14 days to allow formation of colonies. Size and number of colonies were visually inspected by fixing and staining in 0.1% Coomassie Blue. To further validate effects of CAERS and CFEZO extracts on U251 colony formation, we carried out Soft Agar Colony Formation Assay. This assay is used to measure the ability of cells to grow in soft agar in an anchorage-independent manner, which is considered the most stringent assay for detecting malignant transformation of cells. In this assay cells are plated in a soft agar media matrix where they are unable to attach to an underlying substrate. If cells are able to proliferate they will grow in clumps forming colonies. As seen in It is generally believed that the induction of apoptosis is the primary cytotoxic mechanism of phytochemicals and the major goal of cancer chemotherapy is to commit tumor cells to apoptosis following exposure to anticancer agents . To deteNext, cells were treated with increasing concentrations of CAERS and CFEZO alone and in combination for 48 h, and frequency of apoptotic cell death was assessed by light microscopy. As seen under an inverted phase microscope, untreated U251 cells grew well to form confluent monolayer with a homogenous morphology containing lightly and evenly stained nuclei . In contApoptotic cell death is accompanied by the shrinkage and fragmentation of both cells and their nuclei and extensive degradation of chromosomal DNA into nucleosomal units . Therefoμg/mL) for 24 hours resulted in significant DNA damage (P < 0.001) compared with control cells. Furthermore, persistence of DNA damage can be observed after growing cells for 24 hours in CAERS- and CFEZO-free medium. Thus, these independent methods  of assessing apoptosis provided similar results, suggesting that the antiproliferative potentials of CAERS and CFEZO are linked to their abilities to induce apoptosis in U251 cells.DNA fragmentation to yield DNA ladders is a characteristic feature of apoptosis . To examc to the cytoplasm [c in both fractions. The data in c into the cytosol. Anticytochrome oxidase IV (COXIV) (a marker for mitochondria) was not detected in the cytosol, indicating that cytosolic fractions were not contaminated with mitochondrial proteins.Apoptosis is tightly regulated by Bcl-2 family of proteins , executed by caspases and, in most physiological and pathological situations, triggered by mitochondrial pathway with eventual release of mitochondrial cytochrome ytoplasm . Therefoβ-actin, the cytosolic protein loading control, was observed in all cases. To substantiate the above finding, we carried out qRT-PCR analysis to assess expression levels of Bcl-2 and Bax gene products following CAERS and CFEZO treatments. We observed that CAERS and CFEZO treatments downregulated transcript of Bcl-2 and, on the other hand, upregulated transcript of Bax.Next, we determined activation of the cysteine proteases, caspase-3 and -9. Combination therapy most impressively activated caspase-9, the central player of intrinsic pathway, and caspase-3, the executioner caspase . We furtκB is responsible for the transactivation of various target genes that are implicated in cell survival and constitutive activation of proteins in the NF-κB signaling pathway is evidenced in glioblastoma cells [κB and p53 after CAERS and CFEZO treatments. We found that CAERS and CFEZO treatments markedly downregulated the expression level of the NF-κB  and flavonoids (CFEZO), alone and in combination, inhibited cell proliferation and colony formation in U251 cells, in a dose- and time-dependent manner. Significantly, the IC50 values of cell viability assays revealed that a combination of low doses  inhibited proliferation and colony formation in U251 cells and induced apoptosis more effectively than high doses of the single agent. Thus, the chemical entities found in the CAERS and CFEZO formula represent an effective combination for exerting synergistic pharmacodynamic actions and neutralizing the adverse effects and toxicities of specific individual chemicals. Interestingly, CAERS and CFEZO treatments recapitulated their growth-suppression potentiality in the contexts of MCF-7 and HeLa cells indicating that their cytotoxic effect is not cell-type specific. On the other hand, these treatments exerted minor effect on the growth of nonmalignant human fibroblasts HF-5, which raises a possibility that these treatments may selectively target malignant, but not normal, cells. However, the assays herein cannot exclude the possibility that there are tissue-specific differences between HF-5 fibroblasts and glioblastoma U251 cells. A further proof of synergistic action of alkaloids (in R. stricta) and flavonoids  was concluded from the observations of the soft agar colony formation assay. This assay is a sensitive parameter of toxicity because the number of colonies formed is assessed when the cells are in a state of proliferation. Thus, in the present study, we have identified a novel combination of two natural dietary agents, CAERS and CFEZO, which exhibit highly synergistic action to target growth of U251 cells and provide an experimental basis for effective chemotherapy of GBM.Current anticancer chemotherapeutic agents for GBM have not significantly improved the survival of glioblastoma patients during the past ten years . Therefo-MDA-231 , 36. In Accumulating evidences demonstrate that a characteristic feature of primary GBM, as well as several phenotypes of cancers, is resistant to commit apoptotic cell death . At the To further confirm apoptogenic effects of CAERS and CFEZO, we looked for some morphologic and biochemical hallmarks of apoptosis. The hallmarks of apoptosis comprise shrinkage of the cell and the nucleus as well as condensation of nuclear chromatin into sharply delineated masses. Later on, the nucleus progressively condenses and breaks up (karyorrhexis) . Eventuac from the mitochondrial intermembrane space. The release of cytochrome c is controlled by two opposing groups of proteins. These are the prosurvival Bcl-2 protein family  that constrain cytochrome release, while Bax and other proapoptosis proteins precipitate the release, and the Bcl-2 : Bax ratio determines the relative sensitivity or resistance of the cells to various apoptotic stimuli [c is released into the cytosol, it mediates activation of caspase-9, and the latter then activates caspase-3 [c in the cytosolic fraction and its decrease in the mitochondrial fraction after treatment of cells with the CAERS and/or CFEZO extracts, indicating that the mitochondrial cytochrome c released into cytoplasm. Additionally, we found increase in activities of the caspases 9 and 3 and PARP-1 cleavage. Intact PARP-1 can help cells to maintain their viability, but cleavage of PARP-1 facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis. Therefore, cleavage of PARP-1 might be the key for the ultimate apoptotic death of U251 cells induced by CAERS and/or CFEZO treatments. CAERS- and/or CFEZO-induced apoptosis was further confirmed by measuring the levels of Bax and Bcl-2 expression. The analysis revealed an increase in Bax expression and a decrease in Bcl-2 expression indicating that CAERS and/or CFEZO treatments tipped balance of Bax : Bcl-2 ratio in favor of apoptosis. This is an interesting finding, since upregulation of the prosurvival proteins Bcl-2 and Bcl-2XL has been described in recurrent GBMs independent of treatment [Most dietary bioactive agents trigger apoptosis by activation of the intrinsic  apoptotic pathway . A pivot stimuli . Once cyaspase-3 . Caspasereatment  and overreatment . On the reatment , 42. TheκB signaling pathway is a characteristic feature of glioblastoma cells [κBp65, an inducible cytoplasmic pool and a basal pool that drives the transcription of antiapoptotic genes; the presence of NF-κB in the nucleus is critical for the maintenance of a malignant phenotype of GBM cells [κBp65. These findings suggest that CAERS and CFEZO specifically targeted the NF-κBp65 counterpart that drives transcription of antiapoptotic genes, which could be a part of their proapoptotic scenarios to inhibit growth of U251 cells. Although the distinct mechanism underlying NF-κBp65 downregulation necessitates further investigation, it is possible that CAERS and CFEZO treatments provoked ubiquitination-mediated NF-κBp65 degradation events. Recent reports have shown that ubiquitin- and proteasome-dependent degradation of the nuclear NF-κBp65 has been suggested to be a candidate mechanism for degradation of nuclear NF-κBp65 to prevent its excessive activation [κBp65 by other dietary agents has been cited [κBp65 by CAERS and CFEZO was associated with repression of its transcriptional targets, survivin, XIAP, and cyclin D1 [κB [κB and its effectors, survivin and XIAP and cyclin D1, which could be partially responsible for their apoptosis-setting-off activities leading to growth inhibition of U251 cells.Aberrant constitutive activation of the NF-ma cells , 33. In BM cells . The dattivation . Of noteen cited , 45. Sigyclin D1 . These fyclin D1 . In partyclin D1 , 48, knoyclin D1 , and inhyclin D1 . Cyclin n D1 [κB . Cyclin The p53 protein plays a central role in the response to a wide range of cellular stresses including DNA damage. Induction of DNA damage initiates a cascade of signaling leading to p53 activation and subsequent transcriptional activation of p53 response genes , thus provoking cell cycle arrest and/or apoptosis . Mutatioc release, activation of caspase-3 and -9, cleavage of PARP-1, and increasing Bax : Bcl-2 ratio. CAERS and CFEZO treatments downregulated expression level of the nuclear NF-κBp65, which was accompanied by repression of its effectors survivin, XIAP, and cyclin D1. On the other hand, CAERS and CFEZO treatments enhanced expression levels of p53 and its downstream targets, p21 and Noxa. This study provides a useful foundation for studying and developing new antiproliferative substances based on these extracts for the treatment of GBM. Further in vivo studies will validate the role of CAERS and CFEZO as new agents for the chemoprevention of GBM.In conclusion, our current investigation clearly showed that both CAERS and CFEZO extracts acted synergistically to control growth and to induce apoptosis in human GBM cells. Apoptosis was evaluated by flow cytometry and mediated by cytochrome


The aim of this study was to identify the determinants of distance walked in six-minute walk test (6MWD) in patients undergoing cardiac surgery at hospital discharge.The assessment was performed preoperatively and at discharge. Data from patient records were collected and measurement of the Functional Independence Measure (FIM) and the Nottingham Health Profile (NHP) were performed. The six-minute walk test (6MWT) was performed at discharge. Patients undergoing elective cardiac surgery, coronary artery bypass grafting or valve replacement were eligible. Patients older than 75 years who presented arrhythmia during the protocol, with psychiatric disorders, muscular or neurological disorders were excluded from the study.P = 0.001), duration of cardiopulmonary bypass (CPB) (P = 0.001), Functional Independence Measure - FIM (0.004) and body mass index - BMI (0.007) with r = 0.91 and r2 = 0.83 with P < 0.001. The equation derived from multivariate analysis: 6MWD = Surgery (89.42) + CPB (1.60) + MIF (2.79 ) - BMI (7.53) - 127.90.Sixty patients  were assessed. In multivariate analysis the following variables were selected: type of surgery (In this study, the determinants of 6MWD in patients undergoing cardiac surgery were: the type of surgery, CPB time, functional capacity and body mass index. Cardiac surgery is a well-established procedure worldwide due to its safety and effectiveness in treating cardiac patients. Among cardiac surgery, we highlight the CABG and valvar replacement .Despite the undeniable efficiency, recovery of functional capacity of patients in the postoperative period of cardiac surgery is one of the important aspects and discussed in literature . A simplGiven that the use of the 6MWT is growing in population undergoing cardiac surgery, the aim of this study was to identify the determinants of 6MWD at hospital discharge in patients undergoing cardiac surgery. In addition to establishing a reference equation and test its reliability.This is an observational study that aimed to investigate the determinants of 6MWD in patients undergoing cardiac surgery.This study was performed at a tertiary cardiology hospital in Brazil. The patients’ assessments were performed in two stages: preoperatively and at discharge. Data from patients’ records were collected, spirometry and implementation of the Functional Independence Measure (FIM) questionnaire and the Nottingham Health Profile were performed. The 6MWT was performed only at the time of patient’s discharge.Patients undergoing elective cardiac surgery, coronary artery bypass grafting and/or valve replacement were eligible for this study. Patients aged over 75 years; who required more than 24 hours of mechanical ventilation or surgical reintervention, patients who had arrhythmia during mechanical ventilation protocol, patients with psychiatric disorders, neurological or muscular disorders that prevented the completion of the protocol were excluded from the study. Patients were recruited from a tertiary hospital cardiology from December 2011 to August 2013. The patients’ characteristics are listed in Table This study was approved by the Research Ethics Committee of the Federal University of Sergipe under protocol number CAAE-0180.0.107.107-11. All patients signed written informed consent before participation.Age, gender, type of surgery (coronary artery bypass grafting (CABG) or valve replacement (non-CABG)), days of hospitalization in the intensive care unit (ICU), length of hospitalization, duration of mechanical ventilation (MV), cardiopulmonary bypass (CPB) time, quality of life , body mass index (BMI), preoperative left ventricular ejection fraction (LVEF), forced vital capacity (FVC), forced expiratory volume in one second (FEV1), presence of diabetes and hemoglobin dosage.The 6MWT was performed according to American Thoracic Society . A plan Pulmonary function was characterized by spirometry using a portable spirometer manufactured by Clement Clarke ONE FLOW®. The forced vital capacity maneuvers were performed following the technical procedures, criteria for acceptability and reproducibility, as recommended by ATS .Functional capacity was assessed by the Functional Independence Measure (FIM), which is part of the Uniform Data System for Medical Rehabilitation and has been widely used and accepted as a measure of functional assessment  and is vThe FIM has 18 items of assessment and is composed of two fields: motor and cognitive. The motor field is composed of 13 items and divided into four categories: personal care, sphincter control, mobility/transfer, locomotion. The cognitive field consists of 5 items, in two categories: communication and social cognition. For each item is assigned a score ranging from 1 = full assistance, 2 = high assistance, 3 = moderate assistance, 4 = minimal assistance, 5 = supervision, 6 = modified independence and 7 = complete independence. Thus the total score ranges from 18  to 126 (complete independence) ,12.To assess the quality of life we used the Nottingham Health Profile, a generic instrument originally developed to assess quality of life in patients with chronic diseases . This isType of surgery, duration of mechanical ventilation, CPB time, days of hospitalization, days of ICU admission, LVEF, hemoglobin and comorbidities (high blood pressure (hypertension)), diabetes and dyslipidemia).P < 0.20). In multivariate analysis, the non-significant variables were excluded from the model one at a time manually following the criterion of the highest P value. The model was determined when all the variables were presented with a level of significance less than 5% (P < 0.05). The dependent variable was the distance in the six-minute walk test. After the analysis, we establish an equation with the selected variables.Data are presented as mean and standard deviation. We used univariate analysis to select the variables to be used in the multivariate analysis model. For univariate analysis, we consider a level of significance less than 20% , ICU days, days of hospitalization, duration of CPB, duration of MV, pre-LVEF, discharge NHP, discharge motor FIM, total discharge FIM, pre-FVC, discharge FVC, pre-FEV1, discharge FEV1, BMI, presence of diabetes and hemoglobin.The reliability of our equation was assessed using the Bland and Altman representation for comparison of the measured and predicted distance with the equation proposed in a second group of 6 patients, 10% of the sample that generated the equation .Ninety-four patients undergoing cardiac surgery were selected, 34 were excluded due to discharge before the 6MWT, MV time greater than 24 hours, need for reoperation, death and denial Figure . The chaThe 6MWT was well tolerated by all patients and no test was interrupted before completing 6 minutes. The mean 6MWD was 260.20 ± 89.20 meters.P = 0.001), type of surgery (P = 0.037), ICU days (P = 0.037), CPB time (P = 0.13), total FIM at discharge (P = 0.074), BMI (P = 0.20) and presence of diabetes (P = 0.20).Univariate analysis selected the following variables considered in the multivariate analysis: age (P = 0.69), presence of diabetes (P = 0.64) and ICU days (P = 0.99) (in order of exclusion) were excluded from the multivariate analysis model.The following variables, age (P = 0.001), CPB time (P = 0.001), total FIM (0.004) and BMI (0.007), considering r = 0.91 and r2 = 0.83 with P < 0.001.Multivariate analysis selected the following variables: type of surgery  and a higher percentage of risk factors such as hypertension, diabetes, dyslipidemia, history of smoking and alcohol consumption .The mean 6MWD assessed in a second group of patients (n = 6), used to test the reliability of the equation generated was 330.14 ± 55.51 meters, which is 106 ± 13% of predicted value, calculated with our equation. The correlation between predicted and measured values was significant , CPB time, total FIM and BMI.Our study is the first to investigate the determinants of 6MWD in patients undergoing cardiac surgery using noninvasive and easy variables to be collected in clinical practice. Moreover, we were the first to propose and test a predictive 6MWD equation at hospital discharge in patients undergoing cardiac surgery.In our study, we observed that the 6MWD was directly associated with the type of surgery, CPB and the Functional Independence Measure (FIM). Furthermore, the 6MWD was negatively associated with BMI.Our results showed the type of surgery (CABG or non-CABG) as the main predictive variable for the 6MWD. The literature is controversial regarding the influence of the type of surgery and physical capacity in postoperative . Our stuIn our study, the influence of CPB in 6MWD was minimal, but positive, and this may have occurred because of the more severe patients were excluded from the group. There are no records in the studies assessing the influence of CPB on walking capacity literature, but some studies show that CPB does not influence the outcome of pulmonary function and postoperative gasometry ,20.The negative influence of BMI is well established in the field of exercise physiology. Enrigth, et al. . noted tThe influence of gender on the 6MWD has been described in some studies ,21,22, hOther studies showed that spirometric values showed a strong positive correlation with the 6MWD in healthy adults ,25 and iThe combination of independent variables was able to explain 86% of the variability in 6MWD in the total sample, which is very satisfactory in comparison with previously published equations for healthy individuals. A previous study found models that explained 42% and 38% of the variation in 6MWD for healthy men and women, respectively . Other fOur study has some potential limitations such as 6MWD assessment before surgery and did not include other variables such as the characterization of the physical activity level before surgery that could influence the functional status of patients. Moreover, the interpretation of our data should be cautious to represent patients from a cardiology service alone.In this study, the determinants of 6MWD at hospital discharge in patients undergoing cardiac surgery were: the type of surgery, CPB time and the Functional Independence Measure and body mass index. This study generated a reliable equation to predict 6MWD.6MWD: Distance walked in six-minute walk test; 6MWT: Six-minute walk test; BMI: Body mass index; CABG: Coronary artery bypass grafting; CPB: Cardiopulmonary bypass; FEV1: Forced expiratory volume in one second; FIM: Functional independence measure; FVC: Forced vital capacity; ICU: Intensive care unit; LVEF: Left ventricular ejection fraction; MV: Mechanical ventilation; NHP: Nottingham health profile.The authors declare that they have no competing interests.GUO, VOC, LPAC, AAAF, MLCN, WMSJ, TCFC and VJSF conceived and planned the activities that led to the study. GUO and LPAC performed assessments. GUO assessed the data. GUO, VOC, MLCN, WMSJ and VJSF prepared the manuscript. All authors read and approved the final manuscript.