Phosphodiesterase 4 (PDE4) inhibitors increase intracellular cyclic adenosine monophosphate (cAMP), leading to regulation of inflammatory cell functions. Roflumilast is a potent and targeted PDE4 inhibitor. The objective of this study was to evaluate the effects of roflumilast on bronchoconstriction, airway hyperresponsiveness (AHR), and airway inflammation in mild asthmatic patients undergoing allergen inhalation challenge.1 was measured until 7 h post challenge. Methacholine challenge was performed on Days 1 (pre-dose), 13 , and 15 , and sputum induction was performed on Days 1, 13, 14 , and 15.25 subjects with mild allergic asthma were randomized to oral roflumilast 500 mcg or placebo, once daily for 14 days in a double-blind, placebo-controlled, crossover study. Allergen challenge was performed on Day 14, and FEV1 (p = 0.02) and the area under the curve (p = 0.01). Roflumilast had a more impressive effect inhibiting allergen-induced sputum eosinophils, neutrophils, and eosinophil cationic protein (ECP) at 7 h post-allergen , and sputum neutrophils (p = 0.04), ECP (p = 0.02), neutrophil elastase (p = 0.0001) and AHR (p = 0.004) at 24 h post-allergen.Roflumilast inhibited the allergen-induced late phase response compared to placebo; maximum % fall in FEVThis study demonstrates a protective effect of roflumilast on allergen-induced airway inflammation. The observed attenuation of sputum eosinophils and neutrophils demonstrates the anti-inflammatory properties of PDE4 inhibition and supports the roles of both cell types in the development of late phase bronchoconstriction and AHR.NCT01365533ClinicalTrials.gov:  Asthma is characterized by the presence of cough, wheeze, dyspnea, reversible airway obstruction and airway hyperresponsiveness. Eosinophils are cells recognized to be a key feature of allergic asthma , howeverThe intracellular signalling molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), are implicated in the pathophysiology of asthma; they promote smooth muscle relaxation and inhibit inflammation . A novelRoflumilast is a selective inhibitor of the PDE4 isoform which is specific for cAMP degradation, and is expressed in several effector cells central to the pathophysiology of asthma including eosinophils, neutrophils and lymphocytes ,11. As aSubjects with allergic asthma develop an immediate IgE-mediated early asthmatic response (EAR) following inhalation of a sufficient dose of an allergen to which they are sensitized . Up to 51 ) ≥ 70% of predicted and baseline methacholine PC20  ≤ 16 mg/mL. Subjects had no other lung disease, no lower respiratory tract infection or worsening of asthma for 6 weeks before screening, and avoided exposure to sensitizing allergens apart from house dust mite. Subjects were steroid-naïve and used infrequent inhaled beta2-agonist for treatment of asthma. Beta2-agonist and caffeinated beverages were withheld for at least 8 h before laboratory visits. Twenty-five subjects, 10 male/15 female, aged 18-54 years old , and ≥ 15% between 3 and 7 h post allergen (LAR) were randomized to two treatment periods separated by 3-5 weeks. To enter a treatment period, methacholine PC20 was required to be within one doubling dose of that measured during screening, and FEV1 ≥ 70% of predicted. Methacholine challenge and sputum induction were performed in the morning of Day 1 (before dosing), Day 13 , and Day 15 . Allergen challenge was performed on the morning of Day 14, and sputum was induced 7 h post allergen challenge. Blood was collected for pharmacokinetics throughout Day 14.The study was approved by the ethics research board of the respective institutions, and signed informed consent of all subjects was obtained. Screening of subjects was performed over 1 week and included 3 consecutive days for assessment of responses to inhaled allergen challenge  for 14 consecutive days. The first dose of study drug was administered in the laboratory after all study procedures were completed. Subjects self-administered the study drug from Day 2 to Day 12. Doses on Days 13 and 14 were administered in the laboratory approximately 15 minutes before study procedures. Roflumilast 500 mcg was selected because it is well tolerated and effective in asthma control at this dose .20 was measured as described by Cockcroft [1 of at least 20% of the baseline value occurred.Methacholine PCockcroft  using ti1 was required to be within 10% of baseline to proceed with the challenge. Allergen inhalation was performed as described by O'Byrne and colleagues [1 at 10 minutes post allergen was reached. FEV1 was then measured at regular intervals until 7 h after allergen inhalation. Only subjects developing a LAR of ≥ 15% fall in FEV1 during the screening period were eligible to participate in the study. All subjects inhaled the same dose of allergen for each of the two treatment periods. EAR was defined as the largest fall in FEV1 within 2 h after allergen challenge, and LAR was defined as the largest fall in FEV1 between 3 h and 7 h after allergen challenge. The early and late responses area under the curve (AUC) were calculated from 0-2 h and 3-7 h, respectively  by plotting the response using graphics software that calculated the area of the FEV1-time response and expressed as liters by hours (Lxh).FEVlleagues  using thlleagues . During Sputum was induced and processed using the method described by Pizzichini and co-workers . The totBlood was collected from 15 subjects at one trial site for pharmacokinetic assessments on Day 14 of each treatment period. Blood was collected pre-dose and post-dose at 15 min, 30 min, 60 min, 90 min, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h. Venous blood was withdrawn into vacuum blood collection tubes containing lithium heparin, and plasma was separated within 30 min by centrifugation at 1000 g for 10 min in a refrigerated centrifuge. Plasma samples were frozen at -20°C then assayed for roflumilast and roflumilast-N-oxide. Analytical measurements of roflumilast and roflumilast N-oxide were performed by a validated method using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (HPLC-MS/MS) and allowed an LLOQ of 0.1 μg/L for both, roflumilast, and roflumilast N-oxide using 0.4 mL human plasma.20, sputum cell numbers and mediator levels were log-transformed prior to analyses. An ANCOVA adopted for the crossover design was performed to evaluate the effects of treatment and period; fixed factors were age, gender, value at the end of washout, and center. The random factor patient nested in sequence was included in the model. It was assumed that the random errors were independent and identically distributed with mean zero and equal variance.The per-protocol data were analyzed to accommodate the crossover design, thereby removing data from subjects not completing both study periods. Data are presented from 22 subjects as mean ± standard error of the mean (SEM) unless otherwise noted. Methacholine PCTwo subjects withdrew prematurely from the study due to adverse events , and one subject was excluded for using inhaled corticosteroids during the trial. Statistical analyses were carried out on the 22 subjects who completed the study per protocol. Baseline characteristics are shown in Table Roflumilast treatment had a significant effect on the total leukocyte count in sputum, thus cell populations are shown as absolute numbers rather than percentages. After allergen inhalation, with placebo treatment, sputum eosinophils increased from a pre-allergen baseline of 28 ± 6/g to 744 ± 199/g at 7 h and 850 ± 227/g at 24 h post-allergen (both p < 0.0001). Compared with placebo, roflumilast inhibited the allergen-induced increase in sputum eosinophils from a pre-allergen value of 29 ± 12/g to 530 ± 182/g at 7 h (p = 0.015) and 446 ± 120/g (p = 0.051) at 24 h post-allergen . Compared with placebo, roflumilast inhibited the allergen-induced increase in sputum neutrophils from 1.13 ± 0.33 × 103/g pre-allergen to 1.66 ± 0.32 × 103/g at 7 h and 1.61 ± 0.38 × 103/g at 24 h post-allergen   from a pre-allergen baseline of 1.0 ± 1.0/g before challenge, to 10 ± 4.0/g (p < 0.0001) at 7 h, and 8.0 ± 3.0/g p = 0.002) at 24 h post-allergen. Compared with placebo, roflumilast inhibited the allergen-induced increase in sputum MCC from pre-allergen value of 1.0 ± 1.0/g before to 3.0 ± 1.0/g and 4.0 ± 2.0/g at 7 and 24 h post-allergen   Figure .The sputum supernatant was assayed for mediators associated with the activation and degranulation of eosinophils and neutrophils. With placebo treatment, the pre-allergen level of ECP rose significantly from 50.4 ± 13.8 mcg/mL to 283.2 ± 62.0 mcg/mL and 304.5 ± 58.5 mcg/mL at 7 h and 24 h post-allergen (p < 0.001). After roflumilast, this increase was attenuated from pre-allergen value of 45.5 ± 15.0 mcg/mL to 178.2 ± 57.6 mcg/mL and 219.9 ± 72.5 mcg/mL at 7 h and 24 h post-allergen, respectively (both p < 0.02). The moderate increase in neutrophil elastase from 21.9 ± 10.2 mcg/mL pre-allergen to 43.0 ± 13.9 mcg/mL at 24 h post-allergen following placebo treatment was significantly attenuated by roflumilast, from 20.6 ± 9.8 mcg/mL pre-allergen to 21.5 ± 8.6 mcg/mL at 24 h post-allergen (p = 0.0001). Post-allergen levels of myeloperoxidase and IL-8 were consistently lower with roflumilast treatment, however not statistically different than placebo (p > 0.05)  and late AUC 3-7 h -1.19 ± 0.25 Lxh compared to placebo -1.73 ± 0.29 Lxh, (p = 0.013). The maximum change in FEV1 during the early response was -0.87 ± 0.08 L  with roflumilast compared to -0.93 ± 0.10 L  with placebo (p = 0.12). The maximum change in FEV1 during the late response was -0.57 ± 0.09 L  with roflumilast compared to -0.73 ± 0.10 L  with placebo (p = 0.02) , and fell significantly 24 h post-allergen with both placebo and roflumilast . However, roflumilast significantly attenuated the allergen-induced fall in AHR from 5.64 ± 1.26 mg/mL pre-allergen to 3.65 ± 0.81 mg/mL 24 h post-allergen, compared to placebo from 5.39 ± 1.18 mg/mL pre-allergen to 1.80 ± 0.42 mg/mL 24 h post-allergen (p = 0.004).The methacholine PCOne subject under treatment with roflumilast experienced a serious adverse event of depression and was withdrawn from the study. This subject did not have a history of depression. Headache was the most frequently reported adverse event (AE) with roflumilast treatment (4 roflumilast versus 0 placebo), followed by nausea (2 roflumilast versus 0 placebo). The majority of AEs were not considered related to study medication. There were no significant differences between treatments in median values of hematology and blood chemistry variables.Pharmacokinetic profiles were obtained from 15 subjects at Day 14. Following repeated oral doses of roflumilast, estimates of roflumilast and the active metabolite N-oxide roflumilast at Day 14 show maximum plasma concentrations of 7.04 ± 0.65 mcg/L and 26.8 ± 2.59 mcg/L reached at 1.83 ± 0.27 h and 4.37 ± 0.5 h, respectively (Figure The orally active PDE4 inhibitors roflumilast and CDP840, given either as a single dose or regularly are known to inhibit the allergen-induced LAR and AHR -31, and 4, IL-4 and IL-5 [In asthma, there is an increase in the number of inflammatory cells within the airways. It is believed that through the release of their mediators and enzymes -6 these and IL-5 . In keepand IL-5 .1 post-dosing consistent with the lack of direct bronchodilatory activity [We hypothesized that roflumilast would suppress the migration and activation of cells involved in the allergic immune response in the airways following allergen inhalation challenge, and thus reduce the physiological responses of EAR, LAR and AHR. This study demonstrates that roflumilast attenuates allergen-induced physiologic responses in subjects with mild allergic asthma, albeit the effect is small as compared to inhaled corticosteroids . Inhibitactivity .This is the first study to demonstrate inhibition of both allergen-induced airway eosinophilia and neutrophilia with pharmacological intervention, and suggests that the broad anti-inflammatory properties of roflumilast may be effective in attenuating the airway neutrophilic inflammation, which is not well-controlled by corticosteroids.1 : forced expiratory volume in one second; IL: interleukin; LAR: late asthmatic response; MCC: metachromatic cells; PC20 : The provocative concentration of methacholine causing a 20% fall in FEV1; PDE4: phosphodiesterase 4; PK: pharmacokinetics; SEM: standard error of the mean.AE: adverse event; AHR: airway hyperresponsiveness; AUC: area under the curve; cAMP: cyclic adenosine monophosphate; cGMP: cyclic guanosine monophosphate; COPD: chronic obstructive pulmonary disease; EAR: early asthmatic response; ECP: eosinophil cationic protein; FEVThis study was funded by NYCOMED. CSW and DB are employees of NYCOMED. The remaining authors have no conflict of interest to declare.GMG and PO'B designed the study and together with CS, DB and L-PB wrote the manuscript. JC, KJK and MD were responsible for medical procedures. RW, FD, JM, and TS performed study analyses. All authors read and approved the final manuscript.


The precision, accuracy, and average recovery of the method were 1.5–5.3%, 95.4–102.5%, and 96.4% for cortisol, and 1.9–6.0%, 89.2–98.8%, and 79.9% for cortisone, respectively. The method was linear from 1.0 to 500.0 ng/mL (r2 = 0.999) for cortisol and 2.5 to 100.0 ng/mL (r2 = 0.998) for cortisone. The limits of detection (LOD) and quantification (LOQ) were 0.2 and 1.0 ng/mL for cortisol, and 1.0 and 2.5 ng/mL for cortisone, respectively. The average cortisol concentration (133.9 ± 63.7 ng/mL) of samples collected between 9:00 and 11:00 a.m. was higher approximately 4.4 times than that of cortisone (30.5 ± 10.7 ng/mL) (P < 0.0001). The average cortisone/cortisol ratio was 0.225. Therefore, the LC-MS/MS method may be useful for the diagnosis of some adrenal diseases and the assessment of 11β-hydroxysteroid dehydrogenase (11β-HSD) activity in clinical laboratories.A fast, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and then the levels of cortisol and cortisone from sera of healthy adults were determined by the LC-MS/MS method. One hundred  Glucocorticoids are a class of steroid hormones that bind to the glucocorticoid receptor and contribute to the hypothalamic-adrenal-pituitary feedback system. They are part of the feedback mechanism in the immune system that turns immune activity down and therefore used to treat diseases caused by an overactive immune system, such as sepsis , allergiβ-Hydroxysteroid dehydrogenase (11β-HSD) is an enzyme that catalyzes the interconversion of physiologically active 11β-hydroxyl glucocorticoid, cortisol, and inactive 11-keto glucocorticoid, cortisone. Endogenous cortisol is reversibly converted to cortisone by 11β-HSD type 1 [β-HSD type 2 predominantly catalyzes the conversion of cortisol to cortisone mostly in mineralocorticoid target tissues [11D type 1  whereas  tissues –8, and d tissues , 10. Cor tissues . Althoug tissues .Cortisol and cortisone are measured directly from biological samples using immunoassays including radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay (CLIA) . Among tReference values of cortisol or cortisone vary between clinical laboratories due to the use of different analytical methods or in-house methods. Thus, validated assays are needed to measure glucocorticoid hormones accurately in samples originated from human. Although the levels of cortisol and cortisone in serum collected from healthy Japanese subjects between 9:00 and 11:00 a.m. were reported using high-performance liquid chromatography (HPLC) method , no studβ-HSD activity by measuring the concentrations of the two steroids in serum collected from Korean healthy volunteers between 9:00 and 11:00 a.m.In this study, the assay method was validated following the recommendations outlined by the Food and Drug Administration (FDA) of the United States . Evaluatd4, 98%) as an internal standard (IS) was purchased from Cambridge Isotope Laboratories, Inc. . Analytical-grade ammonium acetate (≥98%) and ethyl acetate (99.8%) and activated charcoal were purchased from Sigma. Gibco fetal bovine serum (FBS) was purchased from Life Technologies . HPLC-grade methanol and water were purchased from Fisher Scientific Korea Ltd. . All solvents were filtered through Advantec membranes with 0.45 μm pore size .Standards of cortisol (98%) and cortisone (98%) were purchased from Sigma Chemical Co. . Deuterated cortisol  who had not received any hormone supplementation were recruited. All serum samples were collected at 9:00–11:00 a.m. using SST tubes  and stored at −70°C until assay. Although most of the samples were collected from women, all the samples were studied as recruited, due to no meaningful difference between male and female in the concentrations of cortisol and cortisone . Informed4 were prepared in methanol at a concentration of 100 μg/mL. Working standards were prepared from the stock standards at a concentration of 1 μg/mL. IS was prepared at a concentration of 0.1 mg/mL in methanol. Finally, the calibration standards were prepared in charcoal-stripped 5% FBS at concentrations of 1.0, 5.0, 10.0, 50.0, 100.0, and 500.0 ng/mL for cortisol and 2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 ng/mL for cortisone. All the standards were stored at −20°C until assay. Sample preparation was a modification of two published procedures [μL) was transferred into a glass tube and mixed with 20 μL of working IS. For extraction, 2 mL of ethyl acetate was added into the tube. The tube was vortexed gently on a vortex mixer for 30 s and centrifuged at 3,000 rpm for 5 min. The upper layer was removed and then the lower organic layer was evaporated to dryness under nitrogen gas. No solid-phase extraction was done. The dried extract was reconstituted with 300 μL of methanol, which was transferred to screw-capped injection vial.Stock solutions of cortisol, cortisone, and cortisol-ocedures , 24. Bri i.d., × 50 mm, l. 3 μm particle size) . The injection volume was 5 μL and the oven temperature was 25°C. The mobile phase consisted of 5 mM ammonium acetate and methanol  and was delivered at a flow rate of 0.25 mL/min. Mass spectral detection of positive ions in multiple-reaction monitoring (MRM) mode was performed using an API 4000 triple-quadrupole mass spectrometer  equipped with a Turbo V source and a TurboIonSpray probe. The following m/z MRM transitions were selected: 363.2 → 121.2 for cortisol, 361.2 → 163.2 for cortisone, and 367.1 → 121.1 for cortisol-d4. The declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were optimized at 79 V, 10 V, 33 V, and 6 V for cortisol, 111 V, 10 V, 33 V, and 30 V for cortisone, and 79 V, 10 V, 33 V, and 6 V for cortisol-d4, respectively. Ionspray voltage (IS) and temperature were 5500 V and 500°C, respectively. Collision gas (CAD), curtain gas (CUR), and ion source gases 1 (GS1) and 2 (GS2) were 6, 20, 60, and 45 psi, respectively. Peak areas of each analyte and the corresponding IS were obtained using Analyst 1.5 data processing software .HPLC was performed using an Agilent 1200 series  equipped with an autoinjector and an autosampler. Separations of the steroids were performed on a Capcell Pak MG-II C18 column  ratios >3 and >10, respectively. The limit of quantitation (LOQ) was determined as the lowest concentration for which accuracy was within ±20% and imprecision was within ±15%.Accuracy and precision are defined as the closeness between the concentrations of the analytes in a standard solution or in a spiked sample and the true concentrations and as the reproducibility of the signals observed by different analysis of an aliquot containing the analytes using a standard solution or spiked sample, respectively . The accRecovery is defined as the closeness between the concentration observed by applying the present assay method to a spiked sample and the true concentration spiked to the sample matrix . RecoverInterference studies were performed by spiking a pooled serum with 10 ng/mL of the following compounds: corticosterone, 11-deoxycortisol, progesterone, testosterone, 17-hydroxyprogesterone, dihydrotestosterone, aldosterone, and dehydroepiandrosterone. Interfering peaks at the cortisol and cortisone channels were identified.t-test. Statistical analysis was performed using GraphPad software . P < 0.05 was considered statistically significant.Data processing and graphic presentation were carried out with MS Office Excel 2007 . The concentrations of serum cortisol and cortisone were assessed statistically using the Student d4. The MRM chromatogram of a serum sample from a healthy subject showed that physiological components in the serum did not interfere with the identification and quantification of the analytes x + 0.00783) and 2.5–100.0 ng/mL for cortisone (y = (0.02539 ± 0.00099)x − 0.00784). The weighed (1/X) least-squares determination coefficients were greater than 0.999 for cortisol and 0.998 for cortisone, indicating very good linearity.A simultaneous quantitative assay method for cortisol and cortisone in serum was validated. The calibration curves were linear in the range of 1.0–500.0 ng/mL for cortisol (The LOQ was found to be 1.0 ng/mL (2.75 nmol/L) for cortisol and 2.5 ng/mL (6.87 nmol/L) for cortisone , indicatn = 5) CVs ranged from 2.7 to 4.6 for cortisol and 3.6 to 6.0 for cortisone, while accuracies (% bias) ranged from 95.4 to 102.5 for cortisol and 92.0 to 98.8 for cortisone. Interassay (n = 5) CVs ranged from 1.5 to 4.5 for cortisol and 1.9 to 5.8 for cortisone, while accuracies ranged from 97.3 to 100.4 for cortisol and 89.2 to 96.0 for cortisone. These results indicate that the CVs and the accuracies were within internationally accepted criteria [The precisions  and the accuracies of the LC-MS/MS method were determined by analyzing QC samples at four different concentrations for cortisol and cortisone . Intra-acriteria . The recP < 0.0001) (The mean concentration of cortisol (133.9 ± 63.7 ng/mL) was about 4.4 times higher than that of cortisone (30.5 ± 10.7 ng/mL) at 9:00–11:00 a.m. ( 0.0001) . The meaFurthermore, the concentrations of cortisol and cortisone were not notably changed with increasing age . In a prβ-HSD type 2 at high concentrations of the substrate [β-HSD type 2 that catalyzes the irreversible conversion of active cortisol into inactive cortisone [The concentrations of serum cortisol were compared with those of serum cortisone . The fitubstrate . This reortisone .No significant differences were found between males and females in the cortisone/cortisol ratios. The average ratio of males and females in this study was 0.225 . This reβ-HSD activity in the study of cortisol-cortisone shuttle. Therefore, the LC-MS/MS method could be an alternative method to conventional enzyme immunoassays for the diagnosis of several adrenal dysfunctions in routine clinical laboratories.In this study, the levels of cortisol and cortisone in the sera from healthy Korean subjects were simultaneously measured using a validated LC-MS/MS method after a simple liquid-liquid extraction. To the best of our knowledge, this work is the first report on simultaneous measurement of cortisol and cortisone in the sera from healthy Korean subjects by LC-MS/MS. The present LC-MS/MS method is rapid, sensitive, specific, and robust for the simultaneous measurements of cortisol and cortisone in serum. The method also may be cost-effective compared with the previous reports using any solid-phase extraction cartridge  or onlin


TLR9 is a receptor for oligodeoxynucleotides that contain unmethylated CpG motifs (CpG). Because TLR9 resides in the endoplasmic reticulum during the quiescence state, CpG binding to TLR9 requires membrane trafficking, which includes the maturation of the CpG-containing endosome. In the present study, we examined the role of PIKfyve, a phosphatidylinositol 3-phosphate 5-kinase, in the regulation of TLR9 signaling. The PIKfyve inhibitor YM201636 inhibited co-localization of the CpG-containing endosome with LysoTracker, which stains acidic organelle, and with TLR9. YM201636 increased the co-localization of CpG with the early endosome marker EEA1 but decreased co-localization with the late endosome marker LAMP1. Similar results were obtained in Raw264.7 cells containing shRNA that targets PIKfyve. CpG-mediated phosphorylation but not lipopolysaccharide (LPS)-mediated phosphorylation of IKK, p38 MAPK, JNK and Stat3 was severely impaired by the loss of PIKfyve function. CpG-mediated expression of cytokine mRNA was also decreased in the absence of PIKfyve. These findings demonstrate a novel role of PIKfyve in TLR9 signaling. TLRs recognize specific molecular patterns expressed by microorganisms, which results in host immune responses against pathogens. TLRs can be divided into two groups based on their subcellular localization. TLR 1, 2, 4, 5 and 6 are cell surface receptors and recognize surface structures of pathogens, whereas TLR 3, 7, 8 and 9 reside in intracellular compartments and detect microbial nucleic acids  at 250 V/950 µF . At 24 h after transfection, puromycin (3 µg/ml) was added to the cells, and the incubation was continued to select for resistant cells. Control cells were prepared as described above with the pH1 vector containing a 400-bp stuffer sequence instead of the target sequence.Raw264.7 cells (ATCC) lacking PIKfyve were prepared as follows: Oligonucleotides with sequences targeting the protein were cloned into the pH1 vector downstream of the H1 RNA promoter as described ,17 to ex2 atmosphere. EGFP-TLR9 was transfected by the NeonTM transfection system (Invitrogen) according to the manufacturer’s protocol. Cells were used for microscopic analysis 24-h after the transfection.EGFP-TLR9 was kindly donated by Miyake K. (University of Tokyo) . Raw264.2 for 2 h at room temperature. Microscopic studies were performed using the Keyence BZ-9000 with CFI Plan Apo VC60xH lens . For quantitative analysis, with the exception of the quantification of time-laps imaging, Z-stacks were captured at 1 µm steps over a Z-axis distance of 5 µm  were incubated with LysoTracker Red in the presence or absence of YM201636 for 30 min, added with FITC-CpG, incubated for additional 10 min and finally washed vigorously five times with PBS by pipetting up and down to remove CpG in the medium. Alternatively, Raw264.7 cells transfected with EGFP-TLR9 were incubated with YM201636 for 30 min, added with ROX-CpG, incubated for additional 10 min and washed. The translocation of CpG within the cells was chased up to 30 min at 37°C by time-lapse imaging. For immunostaining, cells were treated with or without YM201636, added with CpG, washed with PBS, and incubated for the indicated periods before being fixed with PBS containing 4% formaldehyde for 15 min. The cells were permeabilized with PBS containing 0.3% Triton X-100 and 0.5% BSA for 60 min, and incubated with anti-LAMP1 or anti-EEA1 at 4 °C overnight. The cells were then incubated with Alexa 647-labeled goat anti-mouse IgG antibody (Fab’) of 5 µm . Stacks 3VO4), 1 mM EDTA, 0.1% BSA, 20 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, 20 µM p-amidinophenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. The cell lysates were centrifuged at 15,000 rpm for 10 min. Supernatants were collected, and the protein concentration was determined using the Bio-Rad assay kit. Total cell lysates (100 µg protein) were mixed with 10 µL of 5x sample buffer (62.5 mM Tris (pH 6.8), 1% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.02% bromophenol blue) and heated at 100 °C for 5 min. The proteins were separated by SDS-PAGE and transferred electrophoretically onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk and incubated with the appropriate antibodies. Antibody binding was detected using a chemiluminescent substrate (PerkinElmer).Cells were washed with PBS and lysed in 50 µL of lysis buffer containing 25 mM Tris-HCl (pH 7.4), 0.5% Nonidet P-40, 150 mM NaCl, 1 mM sodium orthovanadate . Single-stranded cDNA was synthesized with M-MLV reverse transcriptase. The cytokine cDNAs were amplified by a PCR method using the specific primers provided in 2 and the resultant disruption of endosome maturation  ATP as the substrates.(TIFF)Click here for additional data file.


SH-SY5Y cells pretreated with different dosages  of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation.There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages  of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca Regional anesthetics have been used widely in clinical settings and as postoperative analgesics, because of their reduced systemic effects Local anesthetics can cause cell apoptosis, induce the release of reactive oxygen species and lactate dehydrogenase (LDH) 2+ currents, which are further divided into Cav3.1, Cav3.2 and Cav3.3 The main route of extracellular calcium influx into cells is via voltage-dependent calcium channels (VDCCs) 2+ concentration of the cerebellar granule cells treated with kainate from 1543 nM to 764 nM and reduced kainate neurotoxicity. Yagami and colleagues found that S-312-d, another L-type voltage sensitive calcium channel blocker, rescued cortical neurons from apoptosis induced by beta amyloid and human group II A secretory phospholipase A2. The neuroprotective effects of CCB were shown by lowering the intracellular Ca2+ concentration. We conjectured that T-type calcium channels, with the pacemaker-like activity, may be involved with the calcium overload of local anesthetic-induced neurotoxicity. In this study, we hypothesize that neurotoxicity induced by bupivacaine involves T-type calcium channels. Therefore, we employed an in vitro model of cytotoxicity using SH-SY5Y cells treated with bupivacaine. In addition, we monitored the effect of NNC 55-0396 dihydrochloride, a highly selective T-type calcium channel blocker that does not significantly alter currents mediated by other subtypes of calcium channels 2+ ([Ca2+]i), apoptosis, and caspase-3 expression, following bupivacaine treatment.Although calcium channel blockers (CCB) can cause cancer cell growth, they can inhibit the neuronal apoptosis in several neuron injury models The SH-SY5Y cell line was purchased from Shanghai Institutes for Biological Sciences . Goat polyclonal anti-caspase-3 antibody was purchased from Santa Cruz Biotechnology , bupivacaine hydrochloride and NNC 55-0396 dihydrochloride were purchased from Sigma  and Boston Biochem , respectively. Other reagents used in this study were as follows: DMEM/F12 medium and fetal bovine serum , 3--2,5-diphenyl-2-tetrazolium bromide , Quest Fluo-8 AM ester , Hoechst 33258 (Beyotime), annexin V-FITC and propidium iodide , and the LDH cytotoxicity detection kit (Beyotime). All other reagents were from commercial suppliers and of standard biochemical quality.2 incubator at 37°C. The medium was replaced every 2 days.SH-SY5Y cells were cultured in DMEM/F12 medium with 15% (v/v) fetal bovine serum, 100 units/mL of penicillin and 100 µg/mL of streptomycin, and maintained in a humidified 5% COTo investigate the effects of different bupivacaine concentrations on SH-SY5Y cell viability, we treated SH-SY5Y cells with 0.1, 0.5, 0.75, 1, 2, 5, or 10 mM bupivacaine for 24 h. The effects of the different bupivacaine concentrations on SH-SY5Y cell viability were evaluated by the MTT assay.Bupivacaine hydrochloride in powder form was dissolved in medium with serum resulting in the final concentration of bupivacaine being 1 mM, based on the results from the cell viability experiment. Cells were pretreated with 10, 50, or 100 µM NNC 55-0396 dihydrochloride (NNC) 30 min prior to treatment with either the culture medium containing 1 mM bupivacaine or an equivalent amount of medium alone for 6, 12, or 24 h. The experimental groups were: (1) NNC 55-0396 dihydrochloride 100 µM (S+NNC 100 group), (2) bupivacaine (S+B group), (3) bupivacaine+NNC 10 μΜ (S+B+NNC 10 group), (4) bupivacaine+NNC 50 μΜ (S+B+NNC 50 group), (5) bupivacaine+NNC 100 µM (S+B+NNC 100 group), and (6) non-pretreated (S group).3 cells/well with 100 µL culture medium per well. The cells were exposed to either 1 mM bupivacaine or an equivalent amount of medium for 6, 12, or 24 h. MTT (20 µL) was added to each well and incubated at 37°C for 4 h. The optical density of the homogenous purple solution was measured using a spectrophotometer . The control group without bupivacaine treatment was set as 100% cell survival and all other groups were normalized to the corresponding control values.Cell viability was measured using the MTT assay as previously described g for 10 min. The supernatant (100 µL) was transferred to 96-well plates and incubated with the same amount of reaction mixture. LDH activity was determined using a colorimetric assay at an absorbance wavelength of 492 nm and a reference wavelength of 655 nm using a spectrophotometer . Background absorbance from the cell-free buffer solution was subtracted from all absorbance measurements. After removal of the buffer from 12-well plates, 1% (v/v) Triton X-100 solution was applied to the remaining cells. The percentage of LDH released into the incubation buffer was calculated as follows: spontaneously released LDH into the buffer/(spontaneously released LDH into the buffer+intracellular LDH released by Triton X-100).LDH activity was determined using an LDH cytotoxicity detection kit after cells were exposed to 1 mM bupivacaine, or an equivalent amount of medium for 6, 12, or 24 h 2+ ([Ca2+]i) from each group after treatment, with or without drugs for 24 h, was measured with Quest Fluo-8 AM ester. Briefly, a 5 mM stock solution of Quest Fluo-8 AM ester was prepared in high-quality anhydrous DMSO and a 10 µM working solution was prepared in Hanks and HEPES buffer (HHBS). The Quest Fluo-8 AM ester reagent concentration was 5 uM. The cells were incubated with the Quest Fluo-8 AM ester for 20 min at room temperature. Cells were washed twice in HHBS to remove excess probe. The experiments were analyzed at excitation and emission wavelengths of 490 and 525 nm, respectively. To determine either the free calcium concentration in the solution ([Ca2+]i) or the Kd of a single-wavelength calcium indicator, the following equation was used: [Ca2+]i = Kd[F−Fmin]/Fmax−F]. Where F is the fluorescence of the indicator at experimental calcium levels, Fmin is the fluorescence in the absence of calcium and Fmax is the fluorescence of the calcium-saturated probe. The dissociation constant (Kd) is a measure of the affinity of the probe for calcium, which is provided in the kit manual.Cytosolic Ca5 cells/well, with 500 µL culture medium per well. Cells were rinsed with phosphate buffered saline (PBS) and collected. Each pellet was resuspended in 500 µL binding buffer. In addition, 5 µL annexin V-FITC and 5 µL propidium iodide were added to each well. After a 5 min incubation, apoptotic cell death was measured by flow cytometry.After cells were treated as described above for 24 h, the cells were seeded into 24-well plates at a concentration of 5×10Cells in 24-well plates were rinsed 3 times with PBS and stained with Hoechst 33258. Subsequently, the cells were examined and photographed under a fluorescence microscope  with a UV excitation wavelength of 300–500 nm. Apoptotic cells were defined on the basis of nuclear morphology changes: chromatin condensation and fragmentation. The number of apoptotic and normal cells was counted manually by researchers blinded to the treatment schedule. For each well, at least 5 different fields were examined and the apoptosis rate was expressed as the percentage of apoptotic cells to the total number of cells counted.g for 15 min at 4°C. The supernatants were centrifuged at 10 000×g for 1 h at 4°C. The samples were then homogenized in lysis buffer B  Triton X-100, 5.0 mmol/L NaF, and 0.02% (w/v) protease inhibitor cocktail). The samples were centrifuged at 10,000×g for 1 h at 4°C, and the supernatants were used for western blot analysis. Protein concentration was determined using the Bradford method, and protein samples were stored at −80°C. Protein samples were dissolved in 4× sample buffer  Coomassie brilliant blue-G, and 8% (w/v) SDS, pH 6.8), and were subsequently denatured at 95°C for 5 min. Equivalent amounts of protein were separated on a 7.5% (w/v) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were incubated overnight at 4°C with either goat polyclonal anti-caspase-3 antibody or anti-β-actin . The membranes were washed extensively with Tris-buffered saline/Tween-20 and incubated for 2 h in peroxidase-conjugated rabbit anti-goat IgG secondary antibody  at room temperature. The immune complexes were detected by enhanced chemiluminescence, and membranes were then exposed to X-ray film. Quantification of protein bands was conducted by scanning the films and importing the images into Adobe Photoshop software . Scanning densitometry was used for semi-quantitative analysis of the data. Caspase-3 protein was normalized to β-actin levels.Culture flasks or plates were quickly rinsed with chilled PBS. Cells were collected using a plastic cell scraper, removed, and lysed in lysis buffer A , 0.1 mmol/L ethylene glycol tetraacetic acid (EGTA), 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 0.02% (w/v) protease inhibitor cocktail (pH 7.9)). After addition of 90 µL NP-40 (10% (v/v)), samples were shaken for 30 sec and then centrifuged at 800×P<0.05 was considered to be statistically significant.Results are presented as the mean±SD. Factorial design ANOVA  was used to analyze the data from the MTT assay, LDH assay, apoptosis assay and western blot assay. Multiple comparisons tests were performed by LSD. A probability value of Viability of SH-SY5Y cells dose-dependently decreased with increasing concentrations of bupivacaine. Treatment with 0.1, 0.5, 0.75, 1, 2, 5, and 10 mM bupivacaine for 24 h resulted in 58±5%, 47±6%, 39±5%, 27±4%, 15±3%, 5±2%, and 2±1% viability, respectively .Compared with the S group, cell viability of SH-SY5Y cells in the S+NNC 100 group was not significantly different at 6, 12, and 24 h. However, 1 mM bupivacaine caused marked cell injury, and cell viability in the S+B group was 47±7%, 36±5% and 26±5% at 6, 12, and 24 h, respectively. Compared with the S+B group, NNC 55-0396 dihydrochloride pretreatment with the three different concentrations protected SH-SY5Y cells against bupivacaine-induced cell injury at 6, 12, and 24 h. Viability of SH-SY5Y cells treated with NNC 55-0396 dihydrochloride improved to 60±8%, 48±6% and 35±4% in the S+B+NNC 10 group, 70±7%, 61±7%, and 45±4% in the S+B+NNC 50 group, and 67±7%, 62±7% and 46±4% in the S+B+NNC 100 group, respectively. Although there was a significant difference between SH-SY5Y cells in the S+B+NNC 10 group and the S+B+NNC 50 and S+B+ NNC 100 groups, there were no significant differences between SH-SY5Y cells in the S+B+NNC 50 and S+B+NNC 100 groups .SH-SY5Y cells in the S group showed marked LDH release, with extracellular LDH being 8.8±1.9%, 9.2±1.6%, and 10.1±1.2% of total LDH at 6, 12, and 24 h, respectively. In addition, there was no significant change in LDH release in SH-SY5Y cells pretreated with 100 μΜ NNC 55-0396 dihydrochloride for 6, 12, and 24 h. However, incubation with 1 mM bupivacaine increased extracellular LDH release to 20.7±2.1%, 27.1±2.8% and 31.3±2.9% at 6, 12, and 24 h, respectively. Interestingly, SH-SY5Y cells pretreated with NNC 55-0396 dihydrochloride resulted in a reduction in LDH release following bupivacaine treatment. Extracellular LDH at 6, 12, and 24 h was 17.3±1.6%, 22.2±2.7% and 25.3±1.6%, respectively, in the S+B+NNC 10 group; was 14.3±1.8%, 16.7±1.6% and 20.1±1.7% in the S+B+NNC 50 group; and was 13.5±1.9%, 16.5±2.1% and 20.8±1.9% in the S+B+NNC 10 group. Although there was a significant difference between SH-SY5Y cells in the S+B+NNC 10 group and the S+B+NNC 50 and S+B+NNC 100 groups, there were no significant differences between SH-SY5Y cells in the S+B+NNC 50 and S+B+NNC 100 groups .2+]i in SH-SY5Y cells in the S group and S+NNC 100 group was 358±25 nM and 372±32 nM, respectively. However, [Ca2+]i in the S+B group increased dramatically after treatment with 1 mM bupivacaine for 24 h to 715±35 nM. SH-SY5Y cells pretreated with NNC 55-0396 dihydrochloride resulted in a reduction of [Ca2+]i following bupivacaine exposure. [Ca2+]i of the cells in the S+B+NNC 10, S+B+NNC 50 and S+B+NNC 100 groups was 657±29 nM, 619±37 nM and 585±39 nM, respectively  and procaspase-3 (inactive form) were measured. The expression of procaspase-3 in SH-SY5Y cells in the S group and S+NNC 100 group was markedly higher than in the other groups. After treatment with 1 mM bupivacaine for 24 h, the expression of procaspase-3 in SH-SY5Y cells decreased and the expression of caspase-3 dramatically increased. However, NNC 55-0396 dihydrochloride pretreatment prevented the bupivacaine-induced reduction in procaspase-3. Therefore, NNC 55-0396 dihydrochloride pretreatment inhibited caspase-3 cleavage. Although the effects of NNC 55-0396 dihydrochloride were significantly different between SH-SY5Y cells in the S+B+NNC 10 group and the S+B+NNC 50 and S+B+NNC 100 groups, there were no significant differences between SH-SY5Y cells in the S+B+NNC 50 and S+B+NNC 100 groups .Generally, nerve damage resulting from local anesthetic exposure is related to the dose, concentration, and the time of exposure to the local anesthetic 2+ concentration with Fluo-8, with absorption and emission peaks at 490 nm and 514 nm, respectively. They can be excited with an argon ion laser at 488 nm, and their emitted fluorescence increases with increasing concentrations of Ca2+.Compared with Fluo-3 or Fluo-4, Fluo-8 is an excellent probe to use with high sensitivity. In this study, we found intracellular Ca2+ concentrations of SH-SY5Y cells treated with 1 mM bupivacaine for 24 h increased sharply and NNC, inhibited the rise of the intracellular Ca2+ concentration and prevented the apoptosis induced by bupivacaine.In the present study, we detected the intracellular Ca2+ currents, and play an important role in many physiological and pathological processes, such as the regulation of cellular excitability, neurotransmitter secretion and release, motor coordination and function, learning and memory, epilepsy, and neuropathic pain The Cav3 family T-type calcium channels generate low-voltage-activated CaWe found that NNC 55-0396 dihydrochloride protection against bupivacaine-induced apoptosis was dose-dependent. Although 10 μΜ NNC 55-0396 dihydrochloride significantly protected SH-SY5Y cells from 1 mM bupivacaine-induced cell death, the effects of 50 μΜ NNC 55-0396 dihydrochloride were notably enhanced. However, there was no significant difference between 50 μΜ and 100 μΜ NNC 55-0396 dihydrochloride pretreatment, demonstrating that the protection of NNC 55-0396 dihydrochloride exhibited a ceiling effect.One limitation of this study was that NNC 55-0396 dihydrochloride is not subtype specific, and may have acted on Cav3.1, Cav3.2 and Cav3.3, which are all expressed in SH-SY5Y cells In summary, we found that treatment of SH-SY5Y cells with 1 mM bupivacaine for 24 h resulted in apoptosis, activation of caspase-3 and release of LDH. Interestingly, inhibition of T-type calcium channels with NNC 55-0396 dihydrochloride reduced bupivacaine-induced cell death, suggesting a novel role for these calcium channels in local anesthetic toxicity.


Caesalpinia sappan and proven to dose-dependently inhibit both melanogenesis and cellular tyrosinase activity via repressing tyrosinase gene expression in mouse B16 melanoma cells. To our knowledge, sappanone A is the first homoisoflavanone to be discovered with melanogenesis inhibitory activity. Our results give a new impetus to the future search for other homoisoflavanone melanogenesis inhibitors.Homoisoflavanone, sappanone A, was isolated from  Although melanin mainly plays a photoprotective role, the accumulation of abnormal amounts of melanin in different parts of the skin, which results in pigmented patches of skin, can also become an esthetic problem. Therefore, several studies have focused on the inhibition of melanogenesis and the prevention of abnormal pigmentation for cosmetic reasons +; 1H-NMR  δ: 5.39 , 6.37 , 6.59 , 6.82 , 6.92 , 6.93 , 7.62 , 7.81; 13C-NMR  δ:180.8 , 165.3 (C-7), 163.9 (C-8a), 148.0 (C-3′), 146.2 (C-4′), 137.0 (C-9), 130.4 (C-5), 129.5 (C-1′), 127.5 (C-3), 124.2 (C-6′), 118.1 (C-5′), 116.4 (C-2′), 116.0 (C-4a), 111.8 (C-6), 103.5 (C-8), 68.8 (C-2). These data were compared with literature values [Sappanone A: yellow powder; m.p. 221–222 °C; ESI/MS v/v) fetal bovine serum at 37 °C in a humidified, CO2-controlled (5%) incubator. The cells were seeded at an appropriate cell density in a 24-well or a 6-well plate. After 1 day of incubation, the cells were treated with various concentrations of drugs in the absence or presence of a stimulation agent (100 μM of IBMX) for another 2 days. Thereafter, the cells were harvested and used for various assays.Mouse B16 melanoma cells (4A5) were obtained from the Bioresources Collection and Research Center . The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%  for a further 2 h incubation. The MTT solution was then removed, DMSO was added, and the absorbance of the dissolved formazan crystals was determined at 570 nm.g for 15 min, the melanin pellets were dissolved in 1 N NaOH containing 20% DMSO for 1 h at 95 °C. The melanin content was measured by the absorbance at 490 nm.At the end of cell cultivation, the cells were harvested and washed twice with PBS. The pelleted cells were lysed in repeated frozen in lysis buffer containing 20 mM sodium phosphate (pH 6.8) and 1% Triton X-100. After centrifugation at 15,000× l-DOPA. The drug-treated cells were washed with ice-cold PBS and lysed with 20 mM phosphate buffer (pH 6.8) containing 1% Triton X-100 and 1 mM PMSF. Detergent was used to release the membrane-bound tyrosinase from the melanosomes. Cells were then disrupted by freezing and thawing. The lysates were centrifuged at 15,000× g for 15 min. Tyrosinase activity was then determined as follows: 1 mL of the reaction mixture contained 50 mM of phosphate buffer (pH 6.8), 2.5 mM of l-DOPA, and 500 μg of the supernatant protein. After a 15 min reaction at 37 °C, dopachrome formation was monitored by measuring absorbance at 475 nm.Tyrosinase activity in B16 cells was examined by measuring the rate of oxidation of ® Green Master Mix (Applied Biosystems). Total RNA was extracted using an RNeasy® mini Kit  according to the manufacturer’s instructions. The quality of the total RNA sample was evaluated by determining the OD260/OD280 ratio. To prepare a cDNA pool from each RNA sample, total RNA (2 μg) was reverse transcribed at 42 °C for 90 min in the presence of oligo(dT) primers  and reverse transcriptase . The oligonucleotides primers for mouse tyrosinase  and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control  were used. After the initial incubation of 2 min at 50 °C, the cDNA was denatured at 95 °C for 10 min followed by 40 cycles of PCR . All results were obtained from at least three independent experiments. The mRNA level of tyrosinase was normalized using GAPDH as an internal control. The relative tyrosinase mRNA content was calculated by dividing the normalized data by that from the IBMX-stimulated control reaction.Real time qRT-PCR were performed on the ABI 7500 Real Time PCR system  using Fast SYBRt test. A value of p < 0.05 or p < 0.01 was considered to be statistically significant.All of the data in the present study were obtained as averages of experiments that were performed at least in triplicate and are expressed as means ± SD Statistical analysis was performed by the Student’s In conclusion, our results not only demonstrate that sappanone A is the first homoisoflavanone to be discovered with melanogenesis inhibitory activity, but also give a new impetus to the future search for other homoisoflavanone melanogenesis inhibitors.


However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known.p,p′-DDE concentrations for 3 days at 37°C and 5% CO2in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p′-DDE incubation periods were also performed in order to monitor sperm rapid Ca2+ responses. All experiments were repeated on a minimum of five sperm samples from different individuals.Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several 2+ concentration ([Ca2+]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively.All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Cap,p′-DDE rapidly increased [Ca2+]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca2+ medium demonstrated that extracellular Ca2+ influx was responsible for this Ca2+ increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p′-DDE-induced [Ca2+]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p′-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p′-DDE concentrations, suggesting that [Ca2+]i rise may cause premature acrosome reaction (P < 0.05).in vitro study, and caution must be taken when extrapolating the results.This is an p,p′-DDE mechanism specific to sperm is shown in this study. p,p′-DDE was able to induce [Ca2+]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors.A novel non-genomic The study was supported by both the Portuguese National Science Foundation  and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare. A 99.1% chemically pure Fresh normozoospermic sperm samples from both human healthy donors recruited at the Biosciences School, University of Birmingham, and Medical Research Institute, University of Dundee (Ethics number 08/S1402); as well as healthy patients undergoing routine semen analysis or fertility treatments in the Human Reproduction Service at University Hospitals of Coimbra were used accordingly to the proper ethical and Internal Review Board of the participating Institutions. All individuals signed informed consent forms. Samples were obtained by masturbation after 3–5 days of sexual abstinence and seminal analysis was performed according to the World Health Organization guidelines . All sam2, 5.4 mM KCl, 0.81 mM MgSO4, 25.0 mM NaHCO3, 1.0 mM NaH2PO4, 116.4 mM NaCl, 5.5 mM d-glucose, 2.5 mM Na–pyruvate, 41.8 mM Na–lactate and 0.3% (w/v) BSA, for at least 3 h at 37°C under 5% CO2/95% air before starting imaging. [Ca2+]i measurements were performed after loading 4 million/ml sperm with the Ca2+ fluorescent marker Oregon Green BAPTA-1AM  for 1 h at 37°C under 5% CO2/95% air, as described elsewhere /Fbasal] × 100%, where ▵F is % change in intensity at time t, F is fluorescence intensity at time t and Fbasal is the mean basal F established in the beginning of each experiment before application of any stimulus. Each cell was considered to respond when the mean of 10 determinations of normalized F during the exposure period differed significantly from the mean of 10 determinations of normalized F during control (or inhibitor) treatment (P < 0.05). Mean amplitudes and percent responsive cells were calculated for each concentration in each sperm sample analyzed.After liquefaction, spermatozoa were prepared by direct swim-up and allowed to capacitate in a supplemented Earle's balanced salt solution (sEBSS) containing 1.8 mM CaClp,p′-DDE on [Ca2+]i, spermatozoa were exposed to a wide range of concentrations (1 pM–50 µM) diluted in standard sEBSS. To further assess the contribution of the internal Ca2+ stores, similar experiments were performed in a low-Ca2+ sEBSS medium . Finally, inhibition studies were performed using 30 µM mibefradil and 10 µM NNC 55-0396 . Solvent controls were carried out with 0.3% (v/v) DMSO.To evaluate the effect of +-based medium  was used, thus allowing proper recordings of CatSper monovalent currents. 5 µM p,p′-DDE was added at specific time points. All experiments were performed at 25°C.Cells were prepared by swim-up and capacitated as described by ® Sperm Separation Medium, Irvine Scientific, CA, USA) and allowed to capacitate for at least 3 h at 37°C under 5% CO2/95% air. Spermatozoa (10 million/ml) were then exposed to several p,p′-DDE concentrations  for 3 days at 37°C under 5% CO2/95% air in order to mimic the putative continuous exposure to toxicants in the female reproductive tract in vivo. Cells were maintained in a phosphate buffered saline  containing 0.9 mM CaCl2, 0.5 mM MgCl2, 5 mM d-glucose, 1.0 mM Na-pyruvate, 10.0 mM Na-lactate, 0.3% (w/v) BSA and 1% (v/v) penicillin/streptomycin, pH 7.2–7.4, according to our formerly described long-standing culture system (g. Solvent controls were performed by adding 0.3% (v/v) DMSO.After liquefaction, spermatozoa were isolated by density gradient centrifugation  as previously described . Two hunPisum Sativum agglutinin coupled to fluorescein isothiocyanate (PSA-FITC), as described elsewhere . Correlations were performed by the Spearman non-parametric test. Results are expressed as mean% ± SEM. P < 0.05 was considered significant.Statistical analysis was carried out using the SPSS version 19.0 software for Windows . All variables were checked for normal distribution and multiple comparisons were performed by paired in vivo conditions we determined that p,p′-DDE did not affect sperm viability, except after 3 days of continuous exposure to the highest p,p′-DDE concentration tested  caused a clear increase in Ca2+ levels within seconds of exposure (when compared with the control), reversible upon sEBSS media washout  caused an immediate decrease in sperm [Ca2+]i that stabilized at a new level within 3 min of exposure and remained unaltered when p,p′-DDE was added . When we analyzed the distribution of the single-cell response we found that 72.2 ± 14.7% of cells responded with an increase in fluorescence intensity of up to 20%, resembling the response observed in spermatozoa exposed from 1 pM to 10 µM p,p′-DDE in standard sEBSS medium. In all these experiments performed in a low-Ca2+ medium, when standard sEBSS was returned to the chamber [Ca2+]i levels increased as expected, and responded normally to the progesterone stimulus  to establish Ca2+-influx and after a delay of 2.5 min the inhibitors were added in separate experiments .10 µM NNC 55-0396 also reversed the p,p′-DDE on [Ca2+]i is mediated by Ca2+ influx and can be reversed by CatSper antagonistic drugs, suggesting that this DDT metabolite activates CatSper. To confirm this, we investigated the effect of 5 µM p,p′-DDE  without changing reversal potential or the characteristic outward rectification of the current , 25 and 50 µM p,p′-DDE significantly reduced acrosomal integrity after 2 days of exposure . This effect was further observed at Day 3 for both 25 and 10 µM p,p′-DDE (P < 0.05). No differences were observed at 1 µM p,p′-DDE in this 3-day long approach. Differences between concentrations were only found at day 3 for 1 and 25 µM p,p′-DDE  were involved, a pharmacological approach was used. In mouse and human sperm, CatSper is believed to be the principal plasma membrane Ca2+ channel  and affect sperm viability long before they reach the oocyte, thus adversely affecting male fertility. p,p′-DDE concentrations in follicular fluid have already been correlated with failed fertilization fertility.In this study, prolonged p,p′-DDE was able to induce a rise in [Ca2+]i in human sperm through a novel non-genomic mechanism involving the opening of the sperm-specific cation channel CatSper and consequently affected acrosome status and sperm survival, ultimately compromising male fertility.Even at concentrations found in reproductive fluids, Supplementary data are available at http://humrep.oxfordjournals.org/.J.R.S., R.S.T., S.J.P., S.M.W. and C.L.B. established the concept and design. R.S.T. and S.M. acquired data. R.S.T., S.J.P. and J.R.S. wrote the paper and all authors contributed to the analysis and interpretation of the results, drafting, revising and approving the article.the Portuguese National Science Foundation (FCT) through a grant attributed to the CNC Institution (PEst-C/SAU/LA0001/2011) and a Wellcome Trust Grant #86470 provided to S.J.P. and C.L.R.B. S.M. was supported by the Infertility Research Trust. R.S.T. is the recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). Funding to pay the Open Access publication charges for this article was provided by The Wellcome Trust.This work was supported by None declared.


Chronic hyperglycemia-associated inflammation plays critical roles in disease initiation and the progression of diabetic complications, including Alzheimer’s disease (AD). However, the association of chronic hyperglycemia with acute inflammation of the central nervous system in the progression of AD still needs to be elucidated. In addition, recent evidence suggests that Glucagon-like peptide-1 receptor (GLP-1R) protects against neuronal damage in the brain. Therefore, the neuroprotective effects of the GLP-1R agonist exendin-4 (EX-4) against hyperglycemia/lipopolysaccharides (LPS) damage were also evaluated in this study.Ten days after streptozotocin (STZ) or vehicle (sodium citrate) treatment in mice, EX-4 treatment (10 µg/kg/day) was applied to the mice before intrahippocampal CA1 injection of LPS or vehicle  and continued for 28 days. This study examined the molecular alterations in these mice after LPS and EX4 application, respectively. The mouse cognitive function was evaluated during the last 6 days of EX-4 treatment. The results showed that the activation of NF-κB-related inflammatory responses induced cognitive dysfunction in both the hyperglycemic mice and the mice that received acute intrahippocampal LPS injection. Furthermore, acute intrahippocampal LPS injection exacerbated the impairment of spatial learning and memory through a strong decrease in monoaminergic neurons and increases in astrocytes activation and apoptosis in the hyperglycemic mice. However, EX-4 treatment protected against the cognitive dysfunction resulting from hyperglycemia or/and intrahippocampal LPS injection.These findings reveal that both hyperglycemia and intrahippocampal LPS injection induced cognitive dysfunction via activation of NF-κB-related inflammatory responses. However, acute intrahippocampal LPS injection exacerbated the progression of cognitive dysfunction in the hyperglycemic mice via a large increase in astrocytes activation-related responses. Furthermore, EX-4 might be considered as a potential adjuvant entity to protect against neurodegenerative diseases. In vitro evidence has also demonstrated that chronic hyperglycemia enhances lipopolysaccharide (LPS)-elicited gene expression involved in inflammation and tissue destruction Chronic hyperglycemia-associated inflammation is considered to play critical roles in disease initiation and the progression of diabetic complications including Alzheimer’s disease (AD). Evidence has shown that diabetes mellitus (DM) increases the risk of dementia by 50–100% for sporadic Alzheimer's disease (SAD) and 100–150% for vascular dementia in vitro, including upregulation of NF-κB, IL-1β, iNOS, TLR4, COX1, and COX2 LPS, also known as a bacterial endotoxin, is gram-negative bacterial cell surface proteoglycan and is traditionally used to stimulate an immune response. Microglia stimulated by LPS have been reported to increase many inflammation-related responses AD is a multifactor disorder; therefore, the development of effective interventions for patients with AD has been challenging. Recent study has suggested that EX-4 administered peripherally not only improves metabolic parameters but also induces beneficial effects on cognitive function p<0.001; p<0.001; p<0.001; p<0.01; p<0.001) or saline injection  = 236.3679, p<0.001), at different time points (p<0.001), and an STZ × different time points interaction was observed (p<0.001). In addition, intrahippocampal LPS injection did not change the blood glucose level of mice with normoglycemia or hyperglycemia (p>0.05). However, EX-4 treatment decreased the blood glucose level at day 32  = 51.4120, p<0.001) and day 39  = 101.9126, p<0.001) only in the hyperglycemic mice. There was no interaction of LPS × EX-4 in the normoglycemic or hyperglycemic mice (p>0.05). Therefore, these results show that an acute high dose of STZ induced a chronic hyperglycemic condition, while EX-4 treatment ameliorated hyperglycemia and had no effect on normoglycemia.In order to assess the effects of EX-4 in normoglycemic and hyperglycemic mice treated with an intrahippocampal LPS/saline injection, we set up a mouse treatment timeline, as shown in p<0.001) and day 39  = 6.5411, p<0.01). In addition, there was no effect of EX-4 on the insulin level of the normoglycemic mice (data not shown). Therefore, these results suggested that EX-4 treatment increased the level of insulin in the hyperglycemic mice.We further examined the levels of insulin in the plasma of the mice . EX-4 trp<0.001), day 3  = 57.2786, p<0.001) and day 4  = 102.0365, p<0.001) as compared with the normoglycemic mice . However, post-hoc multiple analysis revealed that the hyperglycemic mice that received an intrahippocampal LPS injection spent significantly more time searching for the platform than those that received saline treatment on training day 4 . First, the effect of hyperglycemia and intrahippocampal LPS injection on spatial reference learning and memory was evaluated. During the training period, the latency to reach the platform in the 4 trials of each training day was averaged and assessed as the spatial learning ability . Our resmic mice . In addi p<0.01; , day 3 . Post-hoc analyses further showed that intrahippocampal LPS injection significantly increased the escape latency in the hyperglycemic mice  = 153.7506,  p<0.01; .p<0.001; p<0.001; p<0.001; During the probe trial, the time spent in the target quadrant was assessed as the retrieval of spatial memory. The time spent in the target quadrant was reduced in the mice that received an intrahippocampal LPS injection  = 22.9610, p<0.001; p<0.01; p<0.001; p<0.01), day 3 (p<0.05) and day 4 (p<0.05) . During the probe trial, EX-4 treatment increased the retrieval of spatial memory in the LPS-injected mice with normoglycemia  = 39.216, p<0.001; p<0.001; The effect of EX-4 treatment on these mice was evaluated at the same time, and the results showed that EX-4 reduced the time searching for the platform in the hyperglycemic mice that received an intrahippocampal LPS injection on training day 2 p<0.001; , day 3 ((p<0.05) . During  p<0.05; . Furtherp>0.05; Furthermore, there was no significant difference in the swimming velocity between these groups of mice p>0.05; . Therefop<0.001), intrahippocampal LPS injection  = 46.3172, p<0.001), EX-4 treatment  = 8.7771, p<0.01), hyperglycemia × intrahippocampal LPS injection  = 5.1498, p<0.05), and hyperglycemia × intrahippocampal LPS injection × EX-4 treatment groups  = 4.3447, p<0.05). In addition, intrahippocampal LPS injection also significantly increased the level of IL-1β in the plasma  = 6.0624, p<0.05). However, EX-4 treatment transiently increased the level of IL-1β in the hippocampus of the normoglycemic mice and decreased the level in the hyperglycemic mice (p<0.05). On day 39, IL-1β was not detected in the hippocampus. In addition, IL-1β was not detected in the plasma after EX-4 treatment. These results show that the IL-1β level was transiently increased in the hippocampus of the hyperglycemic mice. In addition, intrahippocampal LPS injection transiently increased the IL-1β levels in the plasma and hippocampus of the mice. Furthermore, intrahippocampal LPS injection enhanced the level of IL-1β in the hippocampus of the hyperglycemic mice. However, EX-4 treatment immediately inhibited the upregulation of IL-1β in the hyperglycemic mice and transiently increased the IL-1β level in the normoglycemic mice that received either an intrahippocampal LPS or a saline injection.The levels of IL-1β in the mouse peripheral and central systems were measured 12 hr after intrahippocampal LPS or saline injection (day 12) and after the MWM (day 39) . On day Molecules related to inflammation and oxidative stress were examined at days 12  and 39 , respectp<0.001; p<0.001; p<0.001; p<0.01; p<0.01; p<0.05; p<0.05; p<0.001; p<0.001; p<0.001; p<0.05; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.01; p<0.05; p<0.001; p<0.05; The results of western blot analysis from day 12 indicated that LPS injection increased the expression levels of TLR4  = 27.5134, p<0.05; p<0.05; p<0.01; p<0.001; p<0.01; p<0.01; p<0.01; p<0.001; p<0.01; p<0.05; p<0.001; p<0.001; p<0.001; p<0.01; p<0.05; p<0.01; p<0.001; After the MWM behavior task (day 39), the results of western blot analysis indicated that LPS treatment significantly increased the levels of COX2  = 8.4511, These results suggested that both chronic hyperglycemia and acute intrahippocampal injection induced the activation of NF-κB-related inflammatory responses. Furthermore, acute intrahippocampal LPS injection did not enhance the activation of the related NF-κB inflammatory responses in the hyperglycemic mice. However, EX-4 treatment not only inhibited the inflammatory-related proteins but also increased the MnSOD level. Therefore, EX-4 treatment induced the effects of both anti-oxidation and anti-inflammation.p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.01; p<0.05; p<0.01; The activation of glia plays an important role in the inflammation of progressive neurodegenerative diseases, including AD Previous studies have shown that many neurochemical abnormalities of the cholinergic, noradrenergic, and serotonergic systems occur in the brains of AD patients p<0.001; p<0.001; p<0.001; p<0.05), intrahippocampal LPS injection × EX-4  = 24.9438, p<0.001), and hyperglycemia × intrahippocampal LPS injection × EX-4  = 13.0401, p<0.001). For 5-HT-ir neurons in the Raphe nucleus, significant differences were also observed due to intrahippocampal LPS injection  = 290.1053, p<0.001; p<0.001; p<0.001; p<0.001), intrahippocampal LPS injection × EX-4  = 41.0257, p<0.001), hyperglycemia × EX-4  = 8.2344, p<0.01), and hyperglycemia × intrahippocampal LPS injection × EX-4  = 123.4330, p<0.001). Therefore, these neurochemical results suggested that acute intrahippocampal LPS injection largely enhanced the deficits in the noradrenergic and serotonergic neurons in the hyperglycemic mice. However, EX-4 treatment protected against the deficits in the noradrenergic neurons of the LC region and the serotonergic neurons of the Raphe nucleus.For the cholinergic neurons, no effect was identified in the mice with hyperglycemia, those that received intrahippocampal LPS injections, and those that received EX-4 treatment in the medial septum, vertical diagonal band of Broca, and horizontal diagonal band of Broca regions (data not shown). For noradrenergic TH-ir neurons in the locus coeruleus (LC), significant differences were observed due to intrahippocampal LPS injection  = 24.1999, p<0.001; p<0.001; p<0.01; p<0.001; p<0.01; We further examined whether neuronal apoptosis was present in the CA1 subregion of the hippocampus of the hyperglycemia or/and an acute intrahippocampal injection groups . The quaIn this study, we aimed to elucidate the mechanisms of chronic hyperglycemia and/or acute intrahippocampal LPS injection in the progression of the cognitive dysfunction. In addition, the neuroprotective effects of the EX-4 against hyperglycemia and/or acute intrahippocampal LPS damage were also evaluated in this study.in vitro study showed that high glucose induced apoptosis through activation of caspase 3 First, mice with STZ-induced diabetes developed hyperglycemia and hypoinsulinemia during the entire experimental period, which was consistent with recent reports Our results also demonstrated that intrahippocampal LPS injection transiently increased the levels of IL-1β in the peripheral/central system and the levels of TLR4, COX1, COX2, CD45, and NF-κB in the hippocampus. In addition, acute intrahippocampal LPS injection resulted in cognitive dysfunction associated with long-term increases in the levels of COX2, CD45, NF-κB, microglia/astrocytes activation, and apoptosis in the hippocampus and decreases in the cognitive-related neurotransmission systems, noradrenergic and serotonergic neurons. Previous study has suggested that IL-1β plays an important role in mediating LPS-induced brain injury Furthermore, we found that intrahippocampal LPS injection transiently enhanced the levels of IL-1β in the hippocampus and chronically exacerbated the impairment of spatial learning and memory associated with highly-induced astrocytes activation and apoptotic levels in the hippocampus and decreased noradrenergic neurons in the LC region and serotonergic neurons in the Raphe nuclei of the hyperglycemic mice. In this study, acute intrahippocmapal LPS injection exacerbated cognitive dysfunction in the hyperglycemic mice. Recent evidence also shows that inflammation induces acute exacerbations of dementia in aged patients During continuous EX-4 treatment, we found that EX-4 decreased blood glucose and increased the insulin level in the hyperglycemic mice, which is consistent with some previous studies In conclusion, our results suggest that the activation of the NF-κB-related inflammatory responses induced cognitive dysfunction in both the hyperglycemia and intrahippocampal LPS injection groups. However, acute intrahippocampal LPS injection exacerbated the progression of cognitive dysfunction through large increases in astrocytes activation-related responses in the hyperglycemic mice. Furthermore, EX-4 treatment protected against the impairment of cognition resulting from hyperglycemia and/or intrahippocampal LPS injection. Therefore, these findings revealed that EX-4 might be considered as a potential adjuvant neuroprotective agent for neurodegenerative diseases.A total of 200 male C57BL/6J mice (6–8 weeks old) were purchased from the National Center for Laboratory Animals Sciences . The mice were randomly divided into eight groups, with 25 animals in each group: (i) normoglycemia/saline vehicle in CA1/saline treatment; (ii) normoglycemia/saline vehicle in CA1/EX-4 treatment; (iii) normoglycemia/LPS in CA1/saline treatment; (iv) normoglycemia/LPS in CA1/EX-4 treatment; (v) hyperglycemia/saline vehicle in CA1/saline treatment; (vi) hyperglycemia/saline vehicle in CA1/EX-4 treatment; (vii) hyperglycemia/LPS in CA1/saline treatment; and (viii) hyperglycemia/LPS in CA1/EX-4 treatment. The mice were housed at 20–25°C and 60% relative humidity under a 12-h light/dark cycle, and food and water were made available ad libitum. All experiments were performed during the light phase between 7 AM and 7 PM. All experimental procedures involving animals were performed according to the guidelines established by the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Taiwan.After one week of adaptation to the home cage, mouse body weight and blood glucose were measured on days 1, 10, 32 and 39 . StreptoMouse blood samples were obtained by tail prick and glucose levels were measured using a commercial glucometer  before hyperglycemia induction. STZ  was intraperitoneally injected into non-fasting mice within 15 min to induce hyperglycemia as previous described After EX-4 or saline intraperitoneal injection, normoglycemic and hyperglycemic subjects were infused with 1 µl of LPS  or vehicle  into the CA1 subregion of the hippocampus. First, each mouse was anesthetized with avertin  and then placed in a stereotaxic instrument . An incision was made in the scalp and a hole was drilled into the skull over the injection site. A 30-gauge-needle was then placed into the dorsal hippocampus. The coordinates for the anterior–posterior (from bregma), medial–lateral (from midline), and dorsal–ventral (from surface of the skull) axes were −2.3, ±2.5, and −1.5 mm, respectively. Bilateral intrahippocampal infusion was administered via a 10.0-µl Hamilton microsyringe with a 30-gauge needle fitted to the arm of the stereotaxic instrument. About 1.0 µl of LPS or vehicle was slowly infused at a rate of 0.35 µl per minute. After an additional 5 min to ensure adequate diffusion, the needle was slowly retracted from the animal. After recovery from surgery, mouse body weight and blood glucose were measured, and a series of cognitive tasks were then performed.Mouse spatial learning and memory were evaluated using a conventional MWM, a device commonly used for studying cognitive deficits in APP transgenic mice Immediately after the water maze test, mice (n = 6 per group) were anesthetized  and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Mouse brains were removed and post-fixed with 4% paraformaldehyde for 4 hr and cryo-protected with 10% sucrose for 1 hr followed by 20% sucrose for 2 hr, then placed in 30% sucrose in PBS for 2 days, followed by continuous serial cryostat sectioning at 30 µm on a crystat-microtome . Histology and immunohistochemistry were performed to assess the location of the infusion needle tip and to identify any anatomical and neurochemical abnormalities induced by the STZ, LPS, and EX-4 treatments. Specific antibodies were used to assess the neurotransmitters by rabbit anti-ChAT polyclonal antibody , rabbit anti-TH polyclonal antibody , and rat anti-serotonin monoclonal antibody . Neuroinflammatory response was identified by rabbit anti-Iba1 polyclonal antibody  and rabbit anti-GFAP polyclonal antibody ; cell apoptosis was analyzed by rabbit anti-caspase3 antibody .2O2. After washing with PBS three times (10 min/wash), nonspecific epitopes of sections were then blocked by incubation in 5% normal goat serum or normal rat serum and 0.1% Triton X-100 in PBS for 1 hr. Sections were incubated with primary antibodies overnight at room temperature, washed with PBS, and incubated in the secondary antibodies  for 1 hr, and finally incubated in an avidin-biotin complex for 1 hr at room temperature. The reaction was developed using a diaminobenzidine (DAB) kit (Vector). All sections were mounted on gelatin-coated slides and cover-slipped for light microscopy. The images obtained were loaded in a research-based digital image analysis software  and the threshold intensity was set manually to keep constant for all photomicrographs as the number of DAB pixels was counted. Pixel counts were taken as the average from three adjacent sections per animal, and the data were presented in Free-floating sections were used for immunohistochemistry and immunofluorescence. In brief, blocking of the endogenous peroxidase of sections was performed by incubation with 3% HProteins were extracted from the whole hippocampus of the mice (n = 6 per group). The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit . Proteins (50 µg) were separated by SDS-PAGE and transferred to membranes. The primary antibodies used were goat anti-TLR4 polyclonal antibody , rabbit anti-MnSOD polyclonal antibody , rabbit anti-NF-κB polyclonal antibody , rabbit anti-CD45 polyclonal antibody , rabbit anti-COX-2 polyclonal antibody , rabbit anti-iNOS polyclonal antibody , mouse anti-COX-1 monoclonal antibody  and mouse monoclonal β-actin antibody ; and the secondary antibodies used were anti-rabbit IgG HRP-linked antibody , anti-goat IgG HRP-linked antibody, and anti-mouse IgG HRP-linked antibody . The specific antibody–antigen complex was detected using an enhanced chemiluminescence detection system . The intensity of the signals of the western blots was quantified using an LAS-3000 chemiluminescence detection system  and was expressed as the ratio relative to β-actin protein.Blood samples were collected from the retro-orbital plexus of anesthetized mice and centrifuged at 2,000×g for 15 min at 4°C on days 12 and 39 (n = 6 for each group). Plasma was collected and stored at −80°C until use. Frozen hippocampus was homogenized and centrifuged as described previously post hoc t-testing in order to compare the effects of all treatments. The results were expressed as the mean ± SEM. Differences were considered statistically significant if p<0.05.Data were analyzed by three-factor analysis of variance (ANOVA) by group (normoglycemia and hyperglycemia), intrahippocampal infusion , and intraperitoneal treatment , followed by Student’s


To examine whether serum uric acid (SUA) is associated with 2-hour postload glucose (2-h PG) in Chinese with impaired fasting plasma glucose (IFG) and/or HbA1c (IA1C).Anthropometric and biochemical examinations, such as SUA concentration, were performed in 3763 individuals from all the villages in Baqiao County, China. A 75-g oral glucose tolerance test (OGTT) was conducted in 1197 Chinese with prediabetes as having IFG (110≤ fasting plasma glucose [FPG] <126 mg/dl and HbA1c <6.5%), IA1C (5.7% ≤ HbA1c <6.5% and FPG <126 mg/dl), or both.P<0.001), 3.06±1.08 (P = 0.001), 5.40±1.26 (P<0.001), and 2.34±2.16 mg/dl (P = 0.056) in all participants, in participants with normal glucose tolerance (NGT), with impaired glucose tolerance (IGT), and with 2-h newly diagnosed diabetes , respectively. In both men and women, 2-h PG increased progressively and significantly from the lower to the upper SUA tertiles (P<0.001). Moreover, in multivariate logistic regression, 1-standard deviation  increment of SUA was significantly associated with a 36% higher risk for 2-h NDM .The present study included 1197 participants with IFG and/or IA1C . In multivariate linear regression, after adjustment for gender, age, smoking and drinking, body mass index (BMI), systolic and diastolic blood pressure , lipid profiles, logarithmic transformed C-reactive protein (log-CRP), estimated glomerular filtration rate (e-GFR), FPG and HbA1c, with a 1-mg/dl increment of SUA, 2-h PG increased by 5.04±0.72 (SUA is significantly associated with 2-h PG in Chinese with IFG and/or IA1C. Serum uric acid (SUA), the end product of purine metabolism, possesses both antioxidant and pro-oxidant properties, which depend on its chemical microenvironment. In clinical investigations, SUA was reported to be associated with gout, hypertension, atherosclerosis, metabolic syndrome, diabetes and prediabetes On the other hand, type 2 diabetes is a recognized and independent risk factor for cardiovascular disease High blood glucose concentration or elevated HbA1c was shown to be a risk factor for cardiovascular complications, even in nondiabetic individuals The present study was conducted from January to May in 2010 in the framework of routine health examinations in all the villages in Baqiao County in Gaoyou, a newly established rural residential area about 299 kilometers northwest to Shanghai, China. Study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. From the local authorities, we obtained the population data of 10008 inhabitants aged from 18 to 74 years. Proportionately stratified random sampling was used to select a representative sample from the total population, and the population size was set at 5000 participants. 3918 individuals, out of the 5000 (78.4%), participated after having given informed written consent. 155 individuals were excluded from the present study, because they had established diabetes , missing information on history of diabetes (n = 17), FPG (n = 18) or HbA1c (n = 8), self-reported gout or using of allopurinol or uricosuric agents (n = 30). After further exclusion of individuals with normal FPG (<110 mg/dl) and HbA1c (<5.7%) (n = 1831) and newly diabetes , 1660 individuals, out of the 3763, with IFG (110≤FPG<126 mg/dl and HbA1c<6.5%), or IA1C (HbA1c: 5.7% ≤ HbA1c<6.5% and FPG<126 mg/dl), or both, were selected and invited for further OGTT, and eventually 1197 individuals with prediabetes as having IFG and/or IA1C participated in the present study.Blood pressure (BP) was measured by trained physician using a mercury sphygmomanometer according to the guidelines of the British Hypertension Society 2) were calculated by using the equation proposed by investigators in the Chronic Kidney Disease Epidemiology (CKD-EPI) Collaboration All laboratory measurements were performed after a fasting of at least 12 h. Plasma glucose (with an intra-assay coefficient of variation [CV] of 2.4% and interassay CV of 3.5%), triglyceride, total and low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol concentrations, serum creatinine, and SUA were measured by enzymatic methods . Hyperuricemia was defined as ≥7 mg/dl (in men) or ≥6 mg/dl (in women). Serum insulin was assessed in duplicate by a highly specific radioimmunoassay using two monoclonal antibodies . C-reactive protein (CRP) was measured by a high-sensitivity turbidimetric immunoassay . Values of estimated glomerular filtration rate . Hepatic insulin sensitivity was evaluated by HOMA with the equation: (fasting glucose [mmol/L] ×fasting insulin [mU/L])/22.5. Whole body Insulin sensitivity index was evaluated by the Matsuda index with the equation: 10,000/square root of (fasting glucose×fasting insulin) × (mean glucose×mean insulin during OGTT). The Matsuda index is strongly related to the euglycemic hyperinsulinemic clamp, the gold standard assessment of insulin sensitivity P<0.05 was considered statistically significant.SAS software, version 9.1 (SAS Institute Inc), was applied for database management and statistical analyses. Skewed variables were log-transformed. Means and proportions were compared by ANOVA and Fisher’s exact test, respectively. Multivariate linear regression was applied to investigate the determinants of 2-h PG in patients with different glucose tolerance  in a full adjustment model including covariables of age, BMI, current smoking and drinking habits, SBP and DBP, lipid parameters, e-GFR, FPG, HbA1c and logarithmic transformed CRP (log-CRP). Multivariate logistic regression was used to study risk factors of 2-h NDM  in a stepwise model, with age, FPG and HbA1C forced in the model. We stratified the participants into three groups by the tertiles of their SUA in men and women, respectively, and investigated their association with 2-h PG by general linear model analysis. Gender interaction analysis was performed by general linear model analysis with gender, SUA and gender*SUA put in the model. Correlations of log-CRP and insulin sensitivity with SUA were studied with Pearson’s method. P = 0.10), DBP (P = 0.05), HDL (P = 0.05), LDL (P = 0.05), creatinine (P = 0.19), and e-GFR (P = 0.52), but a significant difference in gender distribution (P = 0.01). Furthermore, from NGT to 2-h NDM group, a gradual and significant increasing trend was observed in age (P = 0.001), BMI (P<0.001), SBP (P<0.001), total cholesterol (P = 0.04), triglycerides (P<0.001), Log-CRP (P = 0.007), FPG (P<0.001), HbA1c (P<0.001), SUA (P = 0.002) and proportion of hyperuricemia (P = 0.002) and hypertension (P<0.001), but a decreasing trend in the proportion of smoking (P = 0.03).Characteristics of the 1197 patients with IFG and/or IA1C are presented in P<0.001), 3.06±1.08 (P = 0.001), 5.40±1.26 (P<0.001), and 2.34±2.16 mg/dl (P = 0.056) in all participants, in participants with NGT, with IGT, and with 2-h NDM, respectively .In P = 0.03).In P = 0.001) and whole body insulin sensitivity calculated by Matsuda index  (P = 0.084).Of note, SUA was also significantly associated with log-CRP   but not The main finding of this study was that elevated SUA in individuals with prediabetes, defined as IFG and/or IA1c, was significantly associated with higher 2-h PG and risk of type 2 diabetes, independent of FPG, HbA1c and other recognized risk factors.Currently, gout and renal disorders were considered as the main consequences of hyperuricemia, and SUA was recognized as a potential risk factor for hypertension A recent study showed that the prevalence of prediabetes was approximately 15% in China In consistence with our findings, previous studies also indicated the significant association between SUA and insulin resistance predominantly in skeletal muscles The strength of the present study includes: the simultaneous measurement of FPG and HbA1c, no usage of any glucose-lowering or SUA-lowering agents, and the population-based design with relatively large sample size and comprehensive adjustment (including HbA1c and FPG). Several limitations of our study need to be considered. First, the cross-sectional design does not allow any causality analysis. Second, selective bias can be introduced, because individuals with relatively lower glucose or HbA1c levels might have a lower participation rate than those with higher glucose or HbA1c levels. Third, some parameters in the present study were estimated by formula or well-accepted methods, such as estimating GFR by the newly CKD-EPI formula and the whole body insulin sensitivity by the OGTT.In conclusion, SUA in individuals with IFG and/or IA1c is strongly associated with 2-h PG, independent of FPG and HbA1c and other established risk factors, suggesting a significant role of SUA in the deterioration of glucose toleration. Interventional studies are warranted to further investigate the nature of this association.


Resistin was first reported to be an adipocyte-specific hormone, but recent studies have indicated a connection between resistin and reproductive function. However, it is not yet known if resistin is expressed by the ovary and if it can affect steroidogenesis in ovarian follicles from prepubertal pigs.In this study, using real time PCR, immunoblotting, and ELISA, we quantified resistin expression and concentration in maturing ovarian follicles  collected from prepubertal pigs. In addition, the dose-responsive effects of recombinant human resistin  on steroid hormone  secretion in culture medium and steroidogenic enzyme  expression in ovarian follicles were determined.P < 0.05) during follicular growth, with large follicles expressing the highest level of this adipokine. Recombinant resistin also increased P4, A4, and T secretion by up-regulating the steady state levels of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. Recombinant resistin had no effects on E2 secretion and CYP19A1 expression in ovarian follicles.We observed that resistin gene and protein expression increased significantly (Our results show resistin expression in ovarian follicles from prepubertal pigs for the first time. We also show that recombinant resistin stimulates steroidogenesis in ovarian follicles by increasing the expression of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. The presence of resistin in the porcine ovary and its direct effects on steroidogenesis suggest that resistin is a new regulator of ovary function in prepubertal animals. Adipose tissue is known to control many cellular events via endocrine, paracrine, or autocrine mechanisms. Adipose tissue is rich in adipokines, secreted cytokines, and hormones that participate in several physiological and pathological processes such as food intake and metabolic control, diabetes, atherosclerosis, immunity, and reproductive function -3. Leptiin vitro. They also demonstrated resistin expression in the testis to be regulated by luteinizing hormone (LH) and follicle stimulating hormone (FSH), two pituitary hormones. In female rats, resistin expression was up-regulated in adipose tissue from pubertal animals , 0.5% NP-40 [vol/vol], 0.5% SDS [wt/vol], and protease inhibitor). Lysates were cleared by centrifugation at 15,000 × g at 4°C for 30 min, and protein content was determined by a protein assay  using bovine serum albumin (BSA) as a standard. All samples were stored at -20°C until further analysis.Experiment 3 investigated the short-term effects of recombinant human resistin on steroid hormone  secretion and steroidogenic enzyme  expression. After isolation, follicles were cut, using small scissors, to facilitate penetration of the compounds into the tissue and removal oocytes and follicular fluids. Whole ovarian follicles, including theca and granulosa cells were cut to small pieces (2–3 mm) and individually cultured in 24-well plates with M199 medium supplemented with 5% FBS [vol/vol] and increasing concentrations of resistin  at 37°C in a humidified atmosphere containing 5% CO2 [vol/vol]. These doses were chosen based on a previously published study  (TBS-T). Membranes were then incubated overnight at 4°C with anti-resistin (cat. #sc-17575), anti-CYP11A1 (cat. #sc-18040), anti-3βHSD (cat. #sc-30820), anti-CYP17A1 (cat. #sc-46084), anti-17βHSD (cat. #sc-26963), or anti-CYP19A1 (cat. #sc-14244) antibodies  on a shaker. After washing with TBS-T, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody . Immune complexes were detected by chemiluminescence (ECL) using Western Blotting Luminol reagent  and visualized with a Chemidoc™ XRS+ system (Bio-Rad Laboratories). Proteins of interest were quantified using Image Lab™ 2.0 software (Bio-Rad Laboratories). An anti-β-actin antibody (1:3000) was used as a loading control .Immunoblotting was performed as previously described . BrieflyA commercially available human resistin enzyme-linked immunosorbent assay (ELISA)  was used to quantify resistin concentrations in ovarian follicles lysates and follicular fluid. Samples were run in triplicate within the same assay. The sensitivity of the resistin assay was 0.016–1 ng/ml, and the intra- and inter-experimental coefficients of variation were 10% and 15%, respectively.Steroid hormone  levels were determined in conditioned culture media using commercially available ELISA kits . E2 levels were also measured in follicular fluid, and samples were run in duplicate within this assay. The sensitivity of each assay was 0.045 ng/ml for P4, 0.019–10 ng/ml for A4, 0.083–16 ng/ml for T, and 9.7–2000 pg/ml for E2. The intra- and inter-experimental coefficients of variation were 6.99% and 4.34% for P4, 5.6% and 12.1% for A4, 3.28% and 6.71% for T, and 2.71% and 6.72% for E2, respectively. Cross-reactivity between T and A4 was 0.9%, and between T and E2 was <0.1%. Cross-reactivity amongst E2, A4, and T was 0%, but cross-reactivity with estrone was 0.2% based on specification sheets provided by the manufacturer. All other samples were run in quadruplicate within each assay.P < 0.05.Real time PCR, Western blot and ELISA experiments were performed three independent times. In each experiment six ovaries from three different animals were selected. Data were plotted as mean ± S.E.M. All remaining data were shown as the mean ± S.D. Statistical analyses were performed using GraphPad Prism 5 software . Data were analyzed using a one-way analysis of variance (ANOVA) test, followed by Tukey’s honestly significant difference (HSD) test. Statistical significance is indicated by different letters or * P < 0.05) . For positive control we used porcine white adipose tissue . The increase in resistin during follicular growth also correlated with a steady increase in estradiol in follicular fluid collected from SFs, MFs, and LFs  , MFs (0.29 ng/μl protein), and LFs (0.411 ng/μg protein) than in follicular fluid collected from corresponding follicles  .Basal steroid secretion in conditioned culture media collected from SFs was 6.2 ng/ml for P4, 3.3 ng/ml for A4, 7.5 ng/ml for T, and 8.7 ng/ml for E2. Recombinant resistin at increasing concentrations stimulated P4  and T  secretion. Both A4 and E2 in SF conditioned culture media remained unchanged by recombinant resistin .Basal steroid secretion in MF conditioned culture media was 8.7 ng/ml for P4, 2.7 ng/ml for A4, 8.6 ng/ml for T, and 9.5 ng/ml for E2. Recombinant resistin at increasing concentrations stimulated P4 , A4 , and T  secretion. Recombinant resistin had no effect on E2 secretion .Finally, basal steroid secretion in LF conditioned culture media was 10.8 ng/ml for P4, 3.1 ng/ml for A4, 13.1 ng/ml for T, and 11.4 ng/ml for E2. Recombinant resistin at increasing concentrations up-regulated P4 , A4 , and T  secretion. Likewise, E2 secretion remained unchanged by recombinant resistin in LF conditioned culture media  was normalized to the expression of GAPDH. Recombinant resistin at all investigated doses stimulated significantly CYP11A1 and 3βHSD expression in SFs (P < 0.05). Resistin at 1, 10, and 100 ng/ml also increased 17βHSD expression in SFs, but no significant changes in expression were observed for CYP17A1 and CYP19  on steroidogenic enzyme mRNA expression, real time PCR was performed on total RNAs isolated from SFs, MFs, and LFs. The expression of each gene , 3βHSD , CYP17A1 , and 17βHSD  increased significantly in MFs after the addition of recombinant resistin . Recombinant resistin had no effect on CYP19 expression.Likewise, the expression of CYP11A1 , 3βHSD , CYP17A1 (at 100 ng/ml of resistin), and 17βHSD  in LFs ; however, CYP19 expression in LFs remained unchanged by recombinant resistin.Recombinant resistin also up-regulated the expression of P < 0.05).The steady state levels of CYP11A1, 3βHSD, CYP17A1, 17βHSD, and CYP19 were investigated in developing ovarian follicles incubated with recombinant resistin, and representative immunoblots from this experiment are shown in Figure P < 0.05).Resistin at increasing concentrations also stimulated CYP11A1, 3βHSD, CYP17A1, and 17βHSD in MFs  dehydrogenase 1; LF: Large follicle; LH: Luteinizing hormone; MF: Medium follicle; P4: Progesterone; R: Resistin; SF: Small follicle; T: TestosteronThe authors declare that they have no competing interests.AR-M contributed substantially to the conception and design of this study, performed experiments, and interpreted all data. MD performed the ELISA experiments. ELG drafted and revised the manuscript, evaluating it critically for important intellectual content. All authors read and approved the final manuscript.