Autotaxin (ATX) is involved in the synthesis of lysophosphatidic acid. Both have recently been linked to cholestatic pruritus and liver injury. We aimed to investigate whether ATX is an indicator of cholestatic liver injury, health-related quality of life (HRQoL) and prognosis based on a group of 233 patients, 118 with primary biliary cholangitis (PBC) and 115 with primary sclerosing cholangitis (PSC). Patients were followed for 1–60 months, cumulative survival rates were calculated. ATX activity was significantly higher in both groups than in the 103 controls, particularly in patients with cirrhosis and in patients with longer disease duration. Ursodeoxycholic acid (UDCA) non-responders with PBC exhibited increased ATX activity. ATX activity was correlated with liver biochemistry, MELD, Mayo Risk scores and was associated with worse disease-specific HRQoL aspects. In both groups, Cox model analysis indicated that ATX was a negative predictor of survival. Increased ATX levels were associated with a 4-fold higher risk of death/liver transplantation in patients with PBC and a 2.6-fold higher risk in patients with PSC. We conclude that in patients with cholestatic conditions, ATX is not only associated with pruritus but also indicates impairment of other HRQoL aspects, liver dysfunction, and can serve as a predictor of survival. Pruritus is a frequent symptom in chronic cholestatic liver conditions such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Recent studies have identified lysophosphatidic acid (LPA) as a potential mediator of pruritus associated with cholestasisPatients with chronic liver disease suffer not only from pruritus but also from numerous other debilitating symptoms, such as fatigue and depression. A significant proportion of patients with chronic cholestasis report a low quality of lifevs. 7.3 ± 3.4 vs. 2.8 ± 1.4 nmol mL−1 min−1 in patients with PBC, PSC and controls, respectively; P < 0.0001 for all; vs. 7.3 ± 3.4 nmol mL−1 min−1, P < 0.0001; vs. 9.3 ± 3.8 nmol mL−1 min−1, P < 0.01) or patients without liver cirrhosis . In healthy controls, ATX activity was higher in females  and was associated with age . In both cholestatic groups analysed separately, ATX activity was independent from age and gender, even if cirrhotic patients were excluded  and was dependent on patient age . ATX activity was strongly correlated with ATX protein levels  with several laboratory markers of disease severity such as ALP, albumin and haemoglobin . We also detected a significant correlation between ATX activity and the Mayo Risk score in patients with PBC . These correlations remained statistically significant when cirrhotic and non-cirrhotic patients were analysed separately, particularly in non-cirrhotic patients . Significant associations were detected between ATX activity and MELD score . These correlations were also present in non-cirrhotic patients when cirrhotic and non-cirrhotic patients were analysed separately . With respect to single bile acids species in patients with PBC, the strongest correlations in the univariate analysis were obtained for glycine and taurine derivatives of the primary acids chenodeoxycholic acid (CDCA) and cholic acid (CA) (P < 0.0001). Moreover, ATX activity was significantly associated with pruritus intensity as measured by the Itch domain of the PBC-40 and PBC-27 questionnaires .As demonstrated in cid (CA) . In the P < 0.0001) and various bile acids derivatives (P < 0.0001). The correlation between the Itch domain and ATX concentrations was even stronger than in patients in the PBC group .In accordance with these findings in the PBC cohort, we detected significant correlations between ATX activity and total bile acids levels . In contrast to findings in both groups, there was no correlation between ATX activity and total serum bile acids level in healthy controls .Total bile acids concentration was markedly elevated in patients from both groups compared to healthy control patients . ATX activity was associated with scores in the Physical Functioning domain on the generic SF-36 questionnaire. In patients in the PSC group, no associations were detected between ATX activity and HRQoL measures in either the PBC-40/PBC-27 or SF-36 questionnaires, with the exception of the Itch domain.The results of the quality-of-life questionnaires in both groups are presented in vs. 9.6 ± 4.4 nmol mL−1 min−1, P < 0.001; 2 = 6.749, P < 0.01). The receiver operating characteristic (ROC) area under the curve (AUC) indicated that the optimal cut-off of ATX activity for discriminating patients with a higher risk of death/liver transplantation was 11.5 nmol mL−1 min−1, with a sensitivity of 76.2% and a specificity of 68.0% . To investigate whether ATX is an independent predictor of survival, we performed multivariate survival analysis using the Cox regression model. In the first step, we indicated other variables influencing survival in the univariate analysis: UDCA non-response status , presence of cirrhosis (chi2 = 7.417 P = 0.007), Mayo Risk score for PBC  and MELD score . Among these factors, ATX, MELD score and UDCA non-response were the independent variables according to multivariate analysis (Patients with PBC were followed up for 1–58 months (mean follow-up time: 11.5 ± 9.2 months). During this time, a total of 21 (17.8%) patients reached end-points: 18 (15.3%) patients were transplanted, and 3 (2.5%) died. Among them, 17 patients had cirrhosis. The event-free survival rate in the PBC group was 82.2%. The cumulative survival probability is presented in of 68.0% . The Coxanalysis .vs. 6.6 ± 3.0 nmol mL−1 min−1, P < 0.001; 2 = 6.73, P < 0.01). Receiver operating characteristic (ROC) area under the curve (AUC) values indicated that the optimal cut-off of ATX activity with which to distinguish patients with a higher risk of end-points was 7.5 nmol mL−1 min−1, with a sensitivity of 62.5% and a specificity of 66.3% . To investigate whether ATX is an independent predictor of survival, we performed a multivariate survival analysis using the Cox regression model. Firstly, we identified other variables influencing survival in the univariate analysis: the presence of cirrhosis  and MELD score . Among the included variables, multivariate survival analysis using Cox regression revealed the MELD score as the only independent variable in patients with PSC (Patients with PSC were followed up for 1–60 months (mean follow-up time: 12.6 ± 11.8 months). During the follow-up period, a total of 32 (27.8%) patients reached end-points: 28 (24.3%) had liver transplantation, and 4 died (3.5%). Among them, 17 patients had cirrhosis. The event-free survival rate in the PSC group was 72.2%. The cumulative survival probability of this group is presented in of 66.3% . With thwith PSC .The ATX-LPA-axis has been implicated in the occurrence of pruritus during cholestasis and in the presence of liver fibrosis and cirrhosis. This study adds several novel findings of elevated clinical and therapeutic relevance. Our results showed that in chronic cholestasis, ATX activity is strongly correlated not only with itch intensity but also with the impairment of other disease-specific aspects of patients’ quality of life. Interestingly, ATX levels were also associated with the severity of liver disease. In both cohorts, ATX levels were correlated with ALP as well as MELD and Mayo Risk scores. Moreover, ATX levels were higher in patients with liver cirrhosis and a longer duration of the disease. Amongst patients with PBC, UDCA non-response was associated with higher ATX activity. Finally, our study indicated that ATX was a potential predictor of overall survival in patients with cholestatic conditions.Recent studies have described a potential role of the ATX-LPA-axis in the pathogenesis of cholestatic itch. The small molecule LPA was identified as a potent neuronal activator in the sera of cholestatic patients suffering from pruritus910Itch domain of the PBC-40/PBC-27 in both cohorts. This finding confirms the pruritogenic role of LPA in patients with chronic cholestatic conditions. Of interest, ATX activity was also correlated with other disease-specific aspects of HRQoL in the PBC group, namely, Fatigue, Cognitive and Emotional domains; however, no relationship between enzymatic activity and ATX protein levels  was seen for the generic SF-36 questionnaire. Notably, in our previous studies on HRQoL in patients with chronic cholestasis, there were only a few associations between laboratory parameters and quality-of-life aspects in addition to a strong link between ALP and Itch domains15The assessment of the relationship between itch intensity and ATX was previously based on the visual analogue scale and data analysing other disease-related symptoms were lacking. In our study, we analysed in detail the association between ATX and pruritus as well as the links between ATX and other aspects of HRQoL in large, well-defined cohorts of patients with PBC and PSC. We used, for the first time in this context, both the generic questionnaire SF-36, which estimates general patient well-being, as well as the disease-specific assessments PBC-40 and PBC-27. PBC-40 and PBC-27 were designed for the assessment of symptoms more specifically related to PBC, which include pruritus, chronic fatigue and cognitive and emotional decline15In our study, serum ATX was not only associated with indices of patient well-being but was also associated with several laboratory parameters. We noted significant correlations with ALP levels in both groups and with AST levels in the PSC cohort. Higher levels of ATX were correlated with worse liver function  and lower platelet counts in PSC and haemoglobin concentrations in both cohorts. Moreover, ATX activity was significantly correlated with MELD scores in both groups. Interestingly, when we analysed non-cirrhotic and cirrhotic patients separately, most associations remained significant in patients in the early stage of liver disease, but not in patients with cirrhosis. This observation suggests that ATX is a valuable indicator of liver injury even in patients without cirrhosis. However, enzyme activity was also higher in patients with cirrhosis and with a longer duration of the disease in both cohorts. Finally, AUC analysis identified serum ATX as a significant indicator of cirrhosis in both PSC and PBC patients. The only exception was a lack of statistical significance with respect to Child-Pugh scores calculated for cirrhotic patients, which may be due to the small number of subjects in each class.et al., who reported that in patients with liver cirrhosis, serum ATX levels were increased compared to healthy subjects and ATX levels were correlated with Child-Pugh and MELD scores. Furthermore, lower ATX levels were independently associated with a longer overall survival in patientsThe correlation between ATX levels and the severity of liver disease has been recently indicated by Pleli in vitro expression of ATX can be induced by cytokines such as TNF activating the NF-kb pathwayATX mRNA levels were previously measured in various tissues and organs, including the brain, adipose tissue, lung, liver, intestine, kidney, ovary and high endothelial venules. However, the source of the circulating protein under normal conditions has not been identifiedATX expression was also found to be augmented in various malignancies such as lymphoma, malignant melanoma and breast cancerIncreased ATX in serum has also been found in patients with liver diseases such as chronic hepatitis C infection8in vitro study demonstrated that natural bile salts act as partial non-competitive inhibitors of ATX, providing a molecular basis for the association between lysophosphatidic acid signalling and cholestatic disordersThe potential pruritogenic role of ATX can be argued. Pruritogen should be secreted into bile as an interruption of enterohepatic circulation by nasobiliary drainage, but ATX is not present in bileOur study has some limitations. The data were derived from a clinical study, which does not allow us to define the exact molecular mechanisms behind the regulation of ATX expression in cholestatic patients. Moreover, the study pertains to Polish individuals, and our findings certainly require validation in further cohorts. Our findings are focused on short-term and general (but not liver-related) survival. Finally, the quality-of-life tools applied are not specifically designed for use in patients with PSC.In conclusion, our study provides novel clinical evidence of the pruritogenic role of the ATX/LPA axis in the pathogenesis of cholestatic itch. The use of validated HRQoL questionnaires gave us the unique opportunity to detect the major associations between elevated ATX activity and other cholestasis-related symptoms such as fatigue and cognitive and emotional decline. ATX inhibitors or LPA receptor blockers may therefore be of use as therapeutic agents in the treatment of pruritus as well as other incapacitating symptoms related to chronic cholestasis. Moreover, ATX activity is correlated with the severity of cholestatic liver disease measured both by clinical and laboratory parameters as well as Mayo Risk scores. Finally, ATX is a significant predictor of death and liver transplantation. These findings do not undermine the key role of existing clinical parameters but suggest that ATX may represent an additional valuable indicator of hepatic injury and a novel prognostic marker in chronic cholestatic conditions.th revision, 2008). The study was performed according to the STROBE  statement.In this cross-sectional study, we included two independent prospective cohorts of Caucasian patients diagnosed with PBC and PSC. These individuals had been recruited at two centres: the Liver Unit, Pomeranian Medical University in Szczecin, and the Liver and Internal Medicine Unit, Department of General, Transplant and Liver Surgery of the Medical University of Warsaw, from September 2009 to October 2014. Both diseases had been diagnosed according to EASL criteria2 = 8.5, P = 0.004), Child-Pugh score  and Mayo Risk Score . A weak relationship was also detected between UDCA responsiveness and Child-Pugh class . UDCA non-responders did not differ from UDCA responders with respect to gender or MELD score (data not shown).One hundred and eighteen patients with PBC  were studied. Fifty-three (46.1%) subjects with PBC had liver cirrhosis confirmed with either histology or imaging techniques . As shown in One hundred and fifteen patients with PSC  were studied. A diagnosis of IgG4-related cholangitis was excluded based on serum IgG4 levels. Patients with PSC were, as expected, younger than subjects with PBC and included more males. Thirty (26.1%) patients with PSC had also been diagnosed with cirrhosis: 16 (53.3%) were in the Child-Pugh class A group, 12 (40%) were in the Child-Pugh class B group, and 2 (6.7%) were in the Child-Pugh class C group. Parameters of cholestasis were elevated, and MELD scores and Mayo Risk scores in patients with PSC were low. These data are summarized in The number of patients treated at our institutions during the study period determined the size of the groups. Potential confounders or effect modifiers have, to the best of our knowledge, been excluded: none of the included subjects suffered from other conditions that could significantly influence HRQoL such as decompensated diabetes mellitus, renal insufficiency requiring dialyses, malignancy, heart failure ≥ NYHA II, rheumatoid arthritis or asthma. Clinical data, Mayo Risk score for PBC and PSC, and responses to HRQoL questionnaires were collected when the patients agreed to participate in the study. Missing data are indicated if present. Liver biochemistry, ATX activity and protein levels and bile acids concentrations were analysed in serum from subjects after fasting. Samples pertaining to the two cholestatic groups were analysed together in the same batch by laboratory personnel blinded to the clinical data.2, 100 mmol/L of Tris (pH = 9.0), and 0.05% Triton X-100 for 60 minutes at 37 °C. Parallel incubations were performed in the presence and absence of 1 mmol/L of lysophosphatidylcholine (LPC) (14:0). The lysophospholipase activity of ATX was determined by measuring the amount of liberated choline as detected by enzymatic fluorimetry using choline oxidase (2 U/mL), horseradish peroxidase (1.6 U/mL), and homovanillic acid as substrates for peroxidase. After the addition of both enzymes to a buffer , the increase in fluorescence was monitored at 37 °C on a NOVOstar analyser . The (endogenous) amount of choline present in the sample without the addition of LPC was subtracted from the amount measured in the presence of LPC. Inter-assay variance was less than 15%, and intra-assay variance was below 10%.Blood samples were allowed to clot before they were centrifuged at 4 °C, and serum was cryopreserved in aliquots at −80 °C. ATX activity was quantified in diluted sera by an examiner blinded to the clinical and serological groups. Serum samples were incubated with a buffer containing 500 mmol/L of NaCl, 5 mmol/L of MgClATX protein content in serum samples was quantified using an in-house optimized sandwich enzyme-linked immunosorbent assay specific for ATX. Briefly, microtiter plates were coated with an anti-human ATX antibody . Samples and recombinant ATX standards  were diluted in phosphate-buffered saline containing bovine serum albumin and 0.05% Tween-20. Captured antigen was detected with biotinylated anti-human ATX antibody  and streptavidin-horseradish peroxidase using tetramethylbenzidine as the chromogenic substrate. The presence of bilirubin and bile salts in cholestatic samples did not interfere with the quantification of ATX.Blood samples for bile acids determination were collected in EDTA-coated tubes. Immediately after collection, plasma was purified through centrifugation at 4 °C. Subsequently, one volume of formic acid 0.5 M was added to plasma samples. Samples were then frozen and kept at −80 °C until analyses. Further analyses were performed as described in detail elsewhere333435Fatigue, Cognitive, Social-Emotional, Itch and Other Symptoms, with higher scores indicating a worse HRQoL. PBC-27 is a simplified questionnaire for PBCMental Component Summary score and a Physical Component Summary score. Scores range from between 0 and 100, with higher scores indicating a better HRQoLThe HRQoL was assessed with both generic  and disease-specific questionnaires (PBC-40 and PBC-27)P < 0.05) in the univariate analysis as covariates with regard to laboratory parameters, prognostic scales and HRQoL. Multivariate survival analysis was performed using Cox’s regression model. A P value of <0.05 was considered statistically significant.The data are presented as mean ± standard deviation (SD) for continuous variables. The data were analysed using Stat-View-5 Software  and SPSS 20.0  and included Fisher’s exact test and ANOVA when appropriate. Categorical data were compared using Levene’s test for equality of variances, and both pooled-variance and separate-variance t-tests were performed to assess the equality of the mean values. Correlation analyses were performed using the Spearman rank or Pearson’s correlation methods. Survival analysis was performed using the Cox regression model. Death and liver transplantation were considered events. Follow-up time was time until death, liver transplantation or the last contact with the patient. Serum ATX cut-off levels predictive of liver cirrhosis and overall survival in PBC and PSC patients were determined using the receiver operating characteristic (ROC) area under the curve (AUC) analysis. Associations between ATX levels and overall survival hazard ratios (HR) were calculated. To identify independent relationships and adjust the effects of covariates, multiple linear regression analyses were performed including all parameters with significant correlations .


Intravenous methylprednisolone (IVMP) pulse therapy is the first-line treatment for the active phase of moderate to severe Graves' orbitopathy (GO). However, acute and severe liver damage has been reported during and after IVMP therapy. In this retrospective study, we investigated risk factors for liver dysfunction during and after IVMP therapy based on 175 Japanese patients with moderate to severe GO and treated at our center between 2003 and 2011. The results showed that seven patients developed severe liver dysfunction with elevated serum alanine aminotransferase (ALT > 300 U/L). Mild (40–100 U/L) and moderate (100–300 U/L) increases of ALT occurred in 62 patients (35%) and 10 patients (6%), respectively. Liver dysfunction was more frequently observed in males, in patients receiving high-dose methylprednisolone, and patients aged over 50 years. Preexistent viral hepatitis was significantly associated with liver dysfunction (65% in patients positive for hepatitis B core antibody and patients positive for hepatitis C virus antibodies). Our study confirmed the association of liver dysfunction with IVMP during and after treatment. It suggests that, in patients with GO, evaluation of preexisting risk factors—including viral hepatitis—and careful weekly monitoring of liver function during IVMP therapy and monthly thereafter for 12 months are warranted. Intravenous methylprednisolone (IVMP) pulse therapy is the first-line treatment for patients with active-phase moderate to severe Graves' orbitopathy (GO) . IVMP isThe causes of IVMP-associated liver damage are incompletely understood. Thus, the aim of the present study was to investigate the risk factors for liver dysfunction during and after IVMP pulse therapy for GO.This was a retrospective study of 175 Japanese patients with moderate to severe GO who were treated in one center from 2003 to 2013. The mean age of the 118 females and 57 males was 51.7 ± 15.5 years. They had been admitted to our university hospital for GO and were treated with an intravenous injection of 1 g of MP daily for 3 consecutive days per week, repeated for three to six cycles, and followed by a tapering dose of oral prednisolone . The daily dose of MP was reduced to 0.5 g except in cases with optic neuropathy after the recommendation by EUGOGO in 2008 [123I uptake on 123I scintigraphy. Orbitopathy was estimated by ophthalmologists using a modified NOSPECS classification [Thyroid diseases were diagnosed by measuring serum-free triiodothyronine (FT3), free thyroxine (FT4), thyroid-stimulating hormone (TSH), thyroglobulin, anti-thyroglobulin antibody, anti-thyroid peroxidase antibody, and anti-thyrotropin receptor antibodies (TRAbs). TRAbs were measured using three commercial kits: TRAb 1st generation , TRAb 2nd generation, human TRAb  and TSAb (Yamasa TSAb kit), and thyroid fication  and the fication . Graves'Liver function tests were performed once a week during pulse therapy and repeated at every visit thereafter for 1 year. Hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), hepatitis B core antibody (HBcAb), and hepatitis C virus antibody (HCVAb) were measured before pulse therapy. The one patient who was HBsAg-positive consulted with a hepatologist, who prescribed 0.5 mg of entecavir, during and after pulse therapy. In addition, 43 patients were HBcAb-positive and 17 were HCVAb-positive. They likewise consulted with hepatologists before pulse therapy. Serum HBV-DNA was not detected in any patient. HBV-DNA and HCV-RNA were also monitored. Liver dysfunction was classified based on serum alanine aminotransferase (ALT) and total bilirubin levels as mild (ALT: 40–100 U/L), moderate (ALT: 100–300 U/L), or severe .Female and male GO patients significantly differed with respect to age, body mass index (BMI), smoking habits, alcohol habits, and HBcAb positivity before pulse therapy . Human Tt-test, one-way ANOVA, or Mann-Whitney U test for the analysis of continuous variables. The χ2 test or Fisher's exact probability test was used to analyze 2 × 2 or 2 × 4 tables. Multivariate logistic regression analyses were carried out to evaluate the risk factors for liver dysfunction, using exact method . In all tests, a P value < 0.05 was considered to indicate significance.Statistical analysis was performed using JMP Pro software . Data are expressed as the mean ± standard deviation. Statistical comparisons were performed using Student's Increases of ALT during and/or after pulse therapy were detected in 79 patients (45%) . Mild (AP < 0.0012) and in patients over the age of 50 years (P < 0.0009). BMI was significantly higher in patients with mild liver dysfunction than in those without liver dysfunction . Liver dysfunction was not associated with smoking or alcohol habit but it was associated with a high dose of MP . Preexistent viral hepatitis was significantly associated with liver dysfunction during and after pulse therapy (P = 0.0035). HBcAb was positive in 43 GO patients (25%) before pulse therapy. In 28 of them (65%), ALT was significantly increased (P = 0.01125), although in most the increase was mild. HCVAb was positive in 17 GO patients (10%) before pulse therapy. In this group, 11 patients (65%) had increased ALT levels (P = 0.01132).Liver dysfunction occurred more frequently in male patients  were associated with liver dysfunction .Although IVMP pulse therapy is widely used as the first-line treatment for active moderate-to-severe orbitopathy, severe related side effects have been reported, the most common of which is hepatotoxicity. In the recent review by Zang et al. , the morIn our series of 175 patients, seven patients (4.0%) developed severe liver dysfunction. The rate of morbidity was similar to the previous report . The cumKoga et al.  reportedOur study identified risk factors for mild to moderate liver dysfunction during and after IVMP therapy for GO. Among male patients, a mild elevation of ALT was associated with a cumulative dose of IVMP > 8 g; in female patients, a moderate elevation of ALT was associated with age over 50 years. A history of HBV and HCV infection also contributed to a high prevalence of hepatotoxicity, as approximately 25% of our GO patients were HBcAb-positive and 10% were HCVAb-positive before pulse therapy. Within this group, 65% had increased ALT levels during and/or after pulse therapy. Multivariate logistic regression analysis showed that gender, age, and cumulative dose of MP were associated with liver dysfunction. Our study in Japanese patients suggests that viral hepatitis, gender, age, and cumulative dose are predisposing risk factors for hepatotoxicity during and after IVMP therapy. The current study also supports recommendations of a cumulative dose of MP < 8 g. However, as even this dose may not be completely safe, careful monitoring of GO patients receiving IVMP is recommended both during and 12 months after therapy.β-oxidation of fatty acids, with subsequent generation of reactive oxygen species and ATP depletion [Although the mechanisms of mild to severe hepatotoxicity remain unclear, reactivation of viral hepatitis , 10, 11,epletion .Salvi et al.  and MariThe drug-drug interaction may be another possible mechanism of liver dysfunction . None ofThere were several limitations to this study. First, it was retrospective in design. However, it allowed us to assess the effect of single and cumulative doses of IVMP, because in line with the EUGOGO's recommendation we reduced the single dose of IVMP from 1 g to 0.5 g. Another limitation of the study was the small number of patients, which prevented definite conclusions because of the low incidence of severe liver dysfunction. Additionally, no histopathological examinations were done and the effectiveness of IVMP for GO was not evaluated. Therefore, further prospective studies are indicated to assess the hepatotoxicity of IVMP during and after pulse therapy for GO.In conclusion, liver dysfunction is frequently associated with pulse therapy for GO, both during and after treatment. Our study supports the careful evaluation of preexisting risk factors  before initiating IVMP therapy in GO patients. In these patients, strict monitoring of liver function once a week during pulse therapy and every month thereafter for the next 12 months is warranted.


Several retinal ischemic diseases can cause neovascular glaucoma (NVG). Trabeculectomy with mitomycin C (MMC) is a relatively better treatment modality in the management of eyes with NVG than other glaucoma surgeries. The aim of this study was to investigate the factors that may influence the outcome of trabeculectomy with MMC for NVG.Forty-nine NVG eyes from 43 patients  underwent primary trabeculectomy with MMC. The mean follow-up period was 16.8 ± 8.1 months . Twenty-one eyes of 21 patients received intravitreal bevacizumab (IVB) 3.6 ± 1.8 days before trabeculectomy with MMC. A Kaplan-Meier survival-curve analysis was used to summarize the cumulative probability of success. We examined the relationship between the surgical outcome and the following surgical factors: gender, age, history of panretinal photocoagulation, history of cataract surgery, history of vitrectomy, preoperative IVB, NVG in the fellow eye, and postoperative complications  by multivariate analysis.The survival rate was 83.7% after 6 months, 70.9% after 12 months, and 60.8% after 24 months. The Kaplan-Meier survival curves showed no significant difference in the survival rate between the eyes with preoperative IVB (n = 21) and the eyes without preoperative IVB (n = 28) (p = 0.14). The multiple logistic regression analysis showed that postoperative hyphema  was significantly associated with the surgical outcome (p = 0.02).Postoperative hyphema was significantly correlated with the outcome of trabeculectomy for NVG. There was no significant association between preoperative IVB and postoperative hyphema or the results of trabeculectomy. Several retinal ischemic diseases can cause neovascular glaucoma (NVG): proliferative diabetic retinopathy (PDR), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), central retinal artery occlusion (CRAO), and ocular ischemic syndrome (OIS) . PatientNVG is a severe form of glaucoma characterized by neovascularization and the proliferation of fibrovascular tissue in the anterior chamber angle. In the early open-angle glaucoma stage, anti-glaucoma drugs or panretinal photocoagulation (PRP) may be effective. However, as the disease progresses, the proliferative fibrovascular membrane causes angle closure. This stage is not reversible by PRP and is often refractory to anti-glaucoma drugs. Trabeculectomy with mitomycin C (MMC) is a good treatment modality in the management of eyes with NVG -4, but iThe pathogenesis of NVG is related to the production of vascular endothelial growth factor (VEGF) by the underlying ischemic retina, which stimulates neovascularization in the anterior chamber angle ,6. BevacAccording to previous reports, patient factors such as previous PRP  and histWe retrospectively reviewed the medical records of 49 NVG eyes from 43 patients  with the presence of neovascularization in the anterior chamber angle and uncontrolled IOP. All patients underwent primary trabeculectomy with MMC at Kyushu University Hospital, Japan, between January 2008 and July 2010 and could be followed up for more than 6 months. This study was approved by the Ethics Committee of Kyushu University . No patients underwent any other glaucoma surgery before the primary trabeculectomy. The etiology of NVG was PDR (43 eyes), CRVO (2 eyes), and CRAO, BRVO, OIS and uveitis (1 eye each). PRP was performed as much as possible except for patients who could not undergo PRP because of vitreous hemorrhage or corneal edema. Excluding those with a history of ischemic cardiac disease or brain infarction and in poor general status, 21 eyes of 21 patients received IVB (1.25 mg/0.05 mL) at 1 to 7 (3.6 ± 1.8) days before trabeculectomy with MMC after 2009 March.All 43 patients underwent trabeculectomy with MMC. A fornix-based conjunctival flap was made. After hemostasis of the episcleral blood vessels with wet-fluid cautery, a 3 × 3-mm, half-thickness triangle scleral flap was made. Surgical sponges soaked in MMC (0.4 mg/mL) were placed under the conjunctival flap for 3 min, followed by irrigation with 300 mL of physiologic saline. A deep trabecular block was removed and a peripheral iridectomy was performed. The scleral flap was sutured with 10-0 nylon sutures. All patients received topical treatment including 0.5% levofloxacin and 0.1% betamethasone for 1 to 3 months after the operation, and the patients whose IOP was elevated received anti-glaucoma drugs. Postoperatively, argon laser suture lysis was performed, depending on the level of IOP and the condition of the conjunctival bleb formation.The criteria for successful surgery were: IOP < 20 mmHg with or without anti-glaucoma drugs, visual acuity more than light perception, and no additional glaucoma surgery (shunt insertion or cyclophotocoagulation). The following patient data were collected: age, gender, preoperative and postoperative  IOP, lens status, previous surgical ocular history (except for glaucoma surgery), preoperative and postoperative visual acuity, the number of preoperative and postoperative anti-glaucoma drugs the patient had been taking, and postoperative complications.Hyphema, one of the potential postoperative complications of trabeculectomy, was defined as hemorrhage in the anterior chamber to the extent of making niveau, even if only a little. Eyes that showed only suspended red blood cells in the anterior chamber, without making niveau, were not defined as having hyphema. The patients’ visual acuity was measured by assessing their decimal visual acuity, and it was calculated after conversion to the logarithm of the minimum of resolution (logMAR).A Kaplan-Meier survival curve was used to estimate the probability of the success rate. We examined the relationships between the surgical risk factors and the surgical outcome. We considered the following ten possible risk factors for surgical outcome: gender, age, history of previous PRP, history of cataract surgery, history of vitrectomy, concurrent vitrectomy, preoperative IVB, NVG in the fellow eye, postoperative hyphema, postoperative choroidal detachment, and postoperative formation of fibrin. All variables were treated as categorical variables. Each categorical variable was coded as either 1 or 0 depending on the presence or absence of the factor, respectively. Frequencies were compared by chi-square test.We estimated the multivariate odds ratio (OR) and 95% confidence interval (CI) of the following three potential risk factors by using a logistic regression analysis: history of vitrectomy ,18, preoTable Table Figure Figure Even with some therapies, the IOP is often increased to an uncontrolled level, sometimes leading to blindness. According to a systematic review and meta-analysis of comparative studies of two or more surgical techniques (one of which had to be trabeculectomy), including patients with open-angle glaucoma, trabeculectomy still offers the possibility of obtaining excellent IOP control at the long-term follow-up in patients with open-angle glaucoma , whereasSeveral previous studies investigated the success rate of trabeculectomy with MMC for NVG. One study reported that the cumulative probability of the success of trabeculectomy was 67.0% after 1 year and 61.8% after 2 to 3 years . Our surAlthough there was a trend for less failure in the IVB group until postoperative 23 months, preoperative IVB finally did not significantly improve the outcome of trabeculectomy with MMC for NVG in our study. Moreover, it did not decrease the risk of postoperative hyphema. Sugimoto et al. , using rIn contrast, Saito et al. reported that preoperative IVB decreased postoperative hyphema and increased the surgical success rate . The disAnother study demonstrated that NVG eyes with a history of vitrectomy had poor surgical outcomes after trabeculectomy . VitrectTrabeculectomy in NVG patients usually results in frequent postoperative complications and poor surgical outcomes. Hyphema has been described as the most frequent postoperative complication of trabeculectomy in NVG patients . HoweverTNF and IL-1 were reported to be capable of stimulating the proliferation of Tenon’s capsule fibroblasts . MoreoveIn view of these reports, when hyphema occurs after trabeculectomy, increased concentrations of some cytokines may lead to a failure of conjunctival bleb formation. Additionally, blood flow into the trabecular meshwork may lead to a stronger wound-healing response or clog the flow of aqueous humor, but further study is required to test this notion.There are a few reports about reducing the incidence of hyphema following trabeculectomy. Wilson et al. reported that during trabeculectomy, filling the anterior chamber with sodium hyaluronate reduced the incidence of postoperative hyphema for glaucoma patients with primary open-angle glaucoma, chronic angle closure glaucoma, congenital glaucoma, or NVG ; they suIn addition, as a new glaucoma surgery, the Ex-PRESS drainage device was introduced in 2011 as an alternative to trabeculectomy . During In the present study, we found that postoperative hyphema was a significant prognostic factor in trabeculectomy for NVG. There was no clear association between preoperative IVB and postoperative hyphema or the result of trabeculectomy.NVG: Neovascular glaucoma; MMC: Mitomycin C; IVB: Intravitreal bevacizumab; PDR: Proliferative diabetic retinopathy; CRVO: Central retinal vein occlusion; BRVO: Branch retinal vein occlusion; CRAO: Central retinal artery occlusion; OIS: Ocular ischemic syndrome; IOP: Intraocular pressure; PRP: Panretinal photocoagulation; VEGF: Vascular endothelial growth factor; OR: Odds ratio; CI: Confidence interval.The authors declare that they have no competing interests.SN participated in the design of the study, performed the statistical analysis and drafted the manuscript. SY is the main researcher and the corresponding author. SN and RA recorded the data of the patients. MY participated in collecting the data and in the statistical analyses. TK, HE and YO drafted and revised the manuscript. TI participated in the design and final approval of manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/14/55/prepub


In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia. Cordyceps sinensis [Berk.] Sacc. (family Hypocreaceae) and the dead caterpillar of Hepialus armoricanus  whose larva is the primary host of the fungus. Cordyceps is one of the most famous and expensive traditional Chinese medicine and healthy food. It is restrictedly distributed in alpine habitats on the Tibetan Plateau in China. Since Cordyceps has the actions of tonifying kidney, replenishing lung, stanching bleeding and resolving phlegm, it has been historically used in clinic for the treatment of hyposexualities, hyperglycemia, hyperlipidemia, asthemia, respiratory disease, and renal dysfunction, etc.1Cordyceps is a composite consisting of the stroma of the fungus Cordyceps sinensis, Hirsutella sinensis, Cephalospovium sinensis, Mortierella SP and Gliocadium roseum. According to the data from National Bureau of Statistics of China, the annual production of these mycelia was over 3000 tons in 2011, demonstrating wide application and recognized therapeutic effects of these mycelia products.The broad spectrum of pharmacological effects of wild Cordyceps has led to an ever-increasing demand on this herbal medicine whose natural resource is increasingly scarce. To meet the consumption demands of wild Cordyceps, several mycelia products have been developed and manufactured in large quantities by using fermentation technology. To date, five mycelia of different fungus isolated from wild Cordyceps have been approved as drugs by China Food and Drug Administration (CFDA), including As alternatives of wild Cordyceps, mycelia have been substantially investigated for their pharmacological effects, showing that the mycelia could invigorate the lung and nourish kidney, improve the actions of heart and liver, and increase immunity, etc.3456789Isaria sinclairii (the imperfect stage of Cordyceps sinclairii) as a potent immunosuppressive constituentIn 1994, myriocin, a natural sphingolipid (SPL), was isolated from the culture broth of SPLs are a complex family of compounds that share a common structural feature, a sphingoid base backbone which is biosynthesized from serine and a long-chain fatty acyl-CoA, and then converted into ceramides, phosphosphingolipids, glycosphingolipids and other species131517Using the method established in our labTo quantitatively profile the SPLs, a MRM-based method established in our lab was further validated for the quantification of SPLs in wild Cordyceps and its mycelia.2 ≥ 0.9977) over wide dynamic ranges which spanned more than 2 orders of magnitude. The LOD ranged between 0.005 and 3.34 nM, whereas LOQ ranged between 0.0167 and 16.7 nM.As shown in As shown in As shown in As shown in 2 ≥ 0.991.As shown in Cordyceps sinensis and Hirsutella sinensis. In Cordyceps sinensis, sphingoid bases were relatively rich and their total content was higher than that of ceramides, which is a reversed rank order of sphingoid bases and ceramides for the other mycelia. In Hirsutella sinensis, however, the total content of sphingoid bases was notably low whereas the abundance of phosphosphingolipids was relatively high, which led to a reversed rank order of sphingoid bases and phosphosphingolipids for the other mycelia.By using the validated method, totally 101 SPLs in wild Cordyceps and its mycelia were quantitatively profiled. To view the overall distribution of SPLs, the total contents of four classes of SPLs were calculated , which sThe quantitative results also revealed distribution pattern of individual SPLs of wild Cordyceps and its mycelia, which was described by classes as below.Hirsutella sinensis and Gliocadium roseum, in which Sa was more abundant than So and the content of S1P was extremely low . Structurally, sphingoid bases with carbon chain length >= 18 were more abundant than those species with carbon chain length <18 in terms of both number of species and content in all samples. When comparing different samples, the total content of sphingoid bases was found to be highest in Cordyceps sinensis, followed by wild Cordyceps , Cephalospovium sinensis, Mortierella SP, Gliocadium roseum and Hirsutella sinensis. Of note, the total content of sphingoid bases in Cordyceps sinensis was more than 3-folds of that in Hirsutella sinensis.Totally 18 sphingoid bases were quantified, including 10 Sos, 6 Sas, one S1P and one S1Po . Levels Hirsutella sinensis and wild Cordyceps. Comparison of the total content of Cers among samples suggested that Cephalospovium sinensis has the highest content of Cers, followed by other mycelia, whereas wild Cordyceps has the lowest content of Cers.Ceramides were the most structurally diverse species. A total of 51 ceramides were quantitatively determined . It can Cordyceps sinensis, the abundance of SMs (0.69 pmol/mg) was about 14-folds lower than that of PI-Cers (10.32 pmol/mg), while in Cephalospovium sinensis, Gliocadium roseum and Mortierella SP, the total content of SMs (about 1.5–8.3 pmol/mg) was comparable to that of PI-Cers (2.4–10.2 pmol/mg). However, different from both Cordyceps sinensis and Cephalospovium sinensis etc, the level of SMs in Hirsutella sinensis and wild Cordyceps was notably high (about 35–42 pmol/mg), while PI-Cers was much less (about 0.1–1.4 pmol/mg).As shown in Gliocadium roseum of which the HexCer’s abundance was even lower than wild Cordyceps.Glycosphingolipids observed in all samples belong to HexCers. Among which, species with sphingoid base bone of d19:2 accounted for majority of the structures (more than 80%). Totally 8 HexCers were quantified and the results were shown in Hirsutella sinensis was separated from other mycelia at PLS 2 vector, and Cordyceps sinensis, Cephalospovium sinensis and other mycelia were separated at PLS 3 vector. PLS-DA was further performed for wild Cordyceps and its mycelia and the plot was shown in 2Y value of 0.55–0.66 and Q2 value of 0.94–0.97. Based on PLS-DA analysis, the SPLs with VIP ≥ 1 were selected as potential markers and further confirmed by using T-test. Finally, potential markers for the classification of wild Cordyceps and Hirsutella sinensis, Cordyceps sinensis, Cephalospovium sinensis, Mortierella SP, Gliocadium roseum were selected as three SPLs with the highest VIP values, respectively  model was employed to visualize the general classification of samples. As shown in ectively . Signifiectively , in whicCordyceps sinensis and Hirsutella sinensis present quite different profiles as evidenced by particularly high abundance sphingoid bases in Cordyceps sinensis and high level of phosphosphingolipids in Hirsutella sinensis.Our study represents the first report for the quantitative profiling of SPLs in wild Cordyceps and its mycelia. By using the fully validated UHPLC-MS method, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled. The results revealed a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. Among five kinds of mycelia, It offers a robust method for evaluating the quality of wild Cordyceps and its mycelia in the aspect of sphingolipidomes. Based on multivariate analysis, respective sphingolipids were found as potential chemical markers for the differentiation of wild Cordyceps and different mycelia. In addition, our study provided comprehensive chemical evidences for the pharmacological effects and clinical efficacy of wild Cordyceps and its mycelia.Methanol (LC-MS grade), isopropanol (LC-MS grade) and chloroform (HPLC grade) were purchased from Avantor Performance Materials, Lnc. . Formic acid (LC-MS grade), acetic acid (LC-MS grade), ammonium acetate (purity ≥ 98%) and potassium hydroxide  were purchased from Sigma-Aldrich . Distilled water was prepared using a Milli-Q system . An internal standard cocktail  containing C17-sphingosine [So (d17:1)], C17-sphinganine [Sa (d17:0)], C17-sphingosine-1-phosphate [S1P (d17:1)], C12-ceramide [Cer (d18:1/12:0)], C12-sphingomyelin [SM (d18:1/12:0)] and C12-glucosylceramide [GlcCer (d18:1/12:0)] was purchased from Avanti Polar Lipids . SPL standards including Cer (d18:1/6:0), Cer (d18:1/18:0), Cer (18:1/22:0), Cer (d18:1/24:0), SM (d18:1/17:0) and SM (d18:1/24:0) were also obtained from Avanti. So (d14:1), So (d20:1) and SM (d18:1/20:0) were obtained from Matreya LLC . Nomenclature of SPL species is conducted according to LIPIDMAPS (Lipidomics Gateway) nomenclature system. For Cer, HexCer, SM, PI-Cer and MIPC, annotation of sphingoid base backbone denotes number of hydroxyl groups, number of total carbon and number of unsaturation degree . Annotation of N-acyl chain in Cer, HexCer, SM, PI-Cer and MIPC indicates number of total carbon and number of unsaturation degree . For some SMs, annotation of total carbon numbers and total number of unsaturation degree denotes the sum of those of sphingoid base backbone and N-acyl chain.Cordyceps sinensis (n = 5) were from Jiangxi Jiminkexin Pharmaceutical Co., Ltd.; Hirsutella sinensis (n = 5) were from Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd.; Gliocadium roseum (n = 2) were from Hebei Changtian Pharmaceutical Co., Ltd.; Mortierella SP (n = 4) were from Hangzhou Tianyuan Pharmaceutical Co., Ltd. and Datong Liqun Pharmaceutical Co., Ltd.; Cephalospovium sinensis (n = 6) were from Yunnan Baiyao Group Lijiang Pharmaceutical Co. Ltd., Shenyang Dongxin Pharmaceutical Co., Ltd., Hunan Kangerjia Pharmaceutical Co., Ltd., Guizhou Liangji Pharmaceutical Co., Ltd. and Jiangsu Shenhua Pharmaceutical Co., Ltd.Wild Cordyceps (n = 9) were purchased from Qinghai Province and Wai Yuen Tong Medicine Company Limited (Hong Kong) in August 2013. Each wild Cordyceps included the whole wild Cordyceps and two parts cut from the whole wild Cordyceps [fruiting body of wild Cordyceps (n = 9) and sclerotium of wild Cordyceps (n = 9)]. The stock solution of internal standard cocktail (IS 25 μM) was prepared sequentially to afford a series of working solutions . A mixture standard stock solution was prepared by dissolving the nine standards in methanol to a final concentration of 50 μM. This standard stock solution was then diluted to yield a series of working solutions of 0.5, 1, 2, 5, 10, 25 and 50 μM.2O], sequentially. The first two supernatants and the third lower layer were combined and N2 dried. All extracts were reconstituted in 150 μL methanol. All solutions were filtered through a 0.22 μm filter before analysis.Sample solutions were prepared following the procedures developed in our previous study→ product ion), fragmentor voltages, and CE values selected for each individual SPLs were given in An optimized LC-MS condition established in our lab was employed2). The lowest concentration in the calibration curve was further diluted for LOQ and LOD tests. LOD and LOQ were defined as the lowest concentration when signal-to-noise ratios (S/N) of about 3 and 10 was obtained, respectively.Linearity was determined by spiking 6 IS into samples prior to extraction. Triplicates of each IS were prepared and analyzed at no less than nine appropriate concentration levels. Calibration curves were constructed by linear regression. Linearity was verified by correlation coefficients  which were analyzed across the whole analysis process. All validation parameters were evaluated by calculating RSDs.The quantitative accuracy of the method was validated by investigating the linear correlation between the added and determined amount of individual SPLs in samples. In the test, a fixed amount of IS (25 pmol) and the amounts of sphingolipid standard varied from 0 to 500 pmol were spiked into a series of samples prior to extraction. The levels of individual SPLs spiked in samples were determined after removal of the pre-determined levels in samples.The dynamic MRM data were processed with Agilent Mass Hunter Workstation Software. Quantitative results of SPLs in samples were calculated based on below formula: Level (targeted SPL) = 25 pmol × [Area (targeted SPL)/Area (IS)]. The quantitative data of wild Cordyceps and its mycelia were converted into Microsoft Excel format and imported into SIMCA 14.0 software  for multivariate analysis. Variables significantly changed among different samples were selected based on VIP ≥ 1 and the validated by using T-test analysis.How to cite this article: Mi, J.-N. et al. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS. Sci. Rep.6, 20870; doi: 10.1038/srep20870 (2016).


Furthermore, ZWT reduced the level of MDA and increased SOD activity. These results demonstrated that ZWT ameliorated Adriamycin-induced NS in rats possibly by inhibiting Adriamycin-induced inflammation damage, enhancing body's antioxidant capacity, thereby protecting glomerulus from injury.Zhen-wu-tang (ZWT), a well-known formula in China, is widely used to treat chronic kidney diseases. However, very little information on ZWT's mechanism of action is currently available. In this study, we investigated the possible protective role and underlying mechanism of ZWT on nephrotic syndrome (NS) induced by Adriamycin  in rats using biochemical and histopathological approaches. ZWT decreased urine protein excretion and the serum levels of total cholesterol, triglycerides, blood urea nitrogen, and creatinine significantly in diseased rats. A decrease in plasma levels of total protein and albumin was also recorded in nephropathic rats. Pathological results show an improved pathological state and recovering glomerular structure in ZWT treatment groups. ZWT decreased renal IL-8 level but increased renal IL-4 level. In addition, rats subjected to ZWT exhibited less IgG deposition in glomerulus compared with model group. RT-PCR results showed that ZWT decreased the mRNA expression of NF- Nephrotic syndrome (NS) is the triad of proteinuria, low serum albumin, and edema. The disease is seen mostly in boys with predominance in children from the Asian subcontinent . The gloκB, a ubiquitous transcription factor, governs the expression of genes encoding for cytokines, chemokines, growth factors, adhesion molecules, and other factors involved in the immune and inflammatory response. A series of correlations between the activation of NF-κB and onset of both immune- and nonimmune-mediated glomerular injury implicate dysregulation of the NF-κB pathway in its pathogenesis [κB in the pathogenesis of NS. Antioxidant therapy is another important strategy for NS treatment and is considered to be an important pathogenic mechanism. Reactive oxygen species (ROS) are continuously produced physiologically and play an important role in the expression of cell functions such as transmission of impulse information. However, excessive production of ROS would act as mediators of adverse events such as inflammation, necrosis, and apoptosis. In the kidney, ROS are important causes of acute and subacute renal failure in most cases. Also, in certain animal models of NS, superoxide anions and hydroxyl radicals are the main causes of nephrotoxicity [NS has been associated with a wide variety of abnormalities in the immune response, especially in cell mediated immunity which supposed that soluble factors secreted by T cells may form the critical upstream signal for initiating glomerular changes in NS . The occogenesis –10. Thertoxicity . It has toxicity , 13. yang and Li Water, which is recorded in Treatise on Febrile Diseases [Zhen-wu-tang (ZWT) is a classic prescription to treat chronic kidney disease (CKD) with the paths of WenDiseases . As we kDiseases . ZWT hasDiseases . FurtherDiseases . However Aconitum carmichaelii Debx.), Zingiberis Rhizoma Recens , Atractylodis Macrocephalae Rhizoma , Paeoniae Radix Alba , and Poria (sclerotium of Poria cocos (Schw.) Wolf.). The dried raw materials of the herbs were purchased from the Zhixin Medicinal Materials Company, Guangzhou, China . All herbal medicines were authenticated by Professor Qiu-Zhen Zhang at Guangdong Chinese Medicine Museum. To assure the quality control, the materials were validated according to the Chinese Pharmacopeia. Dexamethasone was purchased from the Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, China (Lot. 111001).ZWT is composed of five herbal medicines: Aconiti Lateralis Radix Praeparata  Co., Ltd.ADR was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Lot. 0901E); Urinalysis Reagent Strip from Acon Biotech (Hangzhou) Co., Ltd. (Lot. 201109144); Coomassie (Bradford) Protein Assay Kits from Nanjing Jiancheng Bioengineering Institute (Lot. 20120413 and 20120604); Interleukin-4 and Interleukin-8 ELISA kits for rats from Abcam (HK) Ltd. , Rabbit Anti-Rat IgG/FITC from Beijing Biosynthesis Biotechnology Co., Ltd. (Lot. 990603), SOD and MDA Assay Kits from Nanjing Jiancheng Bioengineering Institute (Lot. 20120523 and 20120324); Trizol , RT Reagent Kit , and SYBR Premix ExTaq II  were from Takara, Dalian, China; the primers for NF- ad libitum to standard laboratory rats chow and water. After 1 week of acclimatization, the rats were divided into five groups : control group; model group; ZWT groups (ADR + ZWT 24.0 g/kg and ADR + ZWT 12.0 g/kg); DXM group (ADR + DXM); the dosages of ZWT were chosen referring to the clinical dosage and the result of our preliminary experiment. The rats were intravenously injected with ADR . The normal group was injected with saline (1.0 mL/100 g) only. One week after injection, the rats were administrated distilled water (control group and model group) or ZWT by an oral gavage method once daily for 28 days. All experiments conformed to the European Community Guidelines and the regulations of the National Institute of Health of US.Fifty adult male special pathogen free Sprague Dawley rats , which weighed (200 ± 20) g, with urine protein qualitative tests showing negative results  (Certificate number SCXK (Guangdong) 2008-0020), were used in the experiments. The rats were housed in an air-conditioned room at 25± 2°C and 65% humidity, with a 12 h light/12 h dark cycle (Certificate number 2008-0085). During the experiments, all animals were givenTo measure urine protein levels, 24 h urine samples were collected using metabolic cages on days 0, 7, 14, 21, 28, and 35. All rats were forbidden from food and free access to water during the course of sample collection. Urinary protein was determined by colorimetric method.On day 35, all animals were sacrificed for blood sample and renal tissue. The blood samples were obtained from abdominal aorta one hour after gavage administration under chloral hydrate anesthesia. Serum was separated by centrifugation at 4°C at 3500 rpm for 15 min and stored at −20°C for biochemical analysis. After exsanguination, the kidneys were rapidly removed and weighted. The two kidneys were individually divided into two parts. The upper pole of the left kidney was treated for light microscopy; the lower pole of the left kidney was divided into four parts, quickly frozen with liquid nitrogen, and kept in −80°C for the assay of IL-4 and IL-8. The upper pole of the right kidney was frozen with liquid nitrogen and then kept in −80°C for immunofluorescence assay of IgG. The lower pole of the left kidney was divided into four parts; one was for electron microscopy, and the rest were quickly frozen with liquid nitrogen and kept in −80°C for gene analysis.Serum cholesterol (CHOL), triglycerides (TRIG), serum albumin (ALB), serum total protein (TP), creatinine (Scr), and blood urea nitrogen (BUN) levels were analyzed using automatic clinical chemistry analyzer. Plasma SOD and MDA levels were tested according to instructions of assay kits. Renal tissue was homogenized in saline (100 mg tissue/mL) with homogenizer and then centrifuged at 4°C at 3000 g for 20 min. The supernatant was kept at −20°C for the assay of interleukin-4 and interleukin-8 levels using ELISA kits.μm sections using freezing microtome and then operated as the following steps: they were fixed in acetone for 10 min, washed in phosphate buffer saline for 3 min, stained with 1 : 50 diluted rabbit anti-rat IgG antibody (FITC labelled) at room temperature for 30 min, washed in phosphate buffer saline twice, 5 min each time, washed in distilled water for 1 min, and wet mounted with 50% glycerin. These finished sections were examined under inverted fluorescence microscopy and eight micrographs were obtained at random with magnification of 400x. The fluorescence signal was quantified using image processing software (Image J 1.47), with eight micrographs for each section and six sections for each group. The result was expressed as average density (/pixel) (average density = integrated density/area).Renal cortical tissues were cut into 5 μm sections and stained with hematoxylin and eosin (H&E) and examined under the light microscope .The cortical tissues were fixed with 10% neutral formalin phosphate buffer, dehydrated through a graded alcohol series, and embedded in paraffin, and then they were cut into 5 A portion of cortical tissues was cut into 1 mm cubes, fixed in 2.5% glutaraldehyde, and postfixed in 1% osmium tetroxide. The samples were dehydrated through a graded alcohol series and embedded in Epon 812. Four ultrathin sections (60 nm) were cut with a diamond knife continuously for each sample and stained with uranyl acetate and lead citrate. The sections were examined under the electron microscope  at 60 kv, ×5000 magnification.κB ; IκB ; GAPDH . The real-time quantitative PCR used ABI7500  and the cycling program was set at 1 cycle of predenaturation at 95°C for 30 s, and then 40 cycles at 95°C for 10 s, 60°C for 34 s, and then melting curve was analyzed. All the RT-PCR experiments were conducted strictly according to the rules of the MIQE.The total RNAs from different experimental groups were obtained by Trizol method. The concentration of RNA was determined by an absorbance at 260 nm and the purity of the RNA was evaluated by measuring the A260/A280 ratio. RNA was reverse transcribed to cDNA using the Takara reverse transcription reagent with gDNA Eraser. All RNA samples performed gDNA removal step . Reverse transcription was performed at 37°C 15 min and 85°C 5 s. PCR was performed with the SYBR Green PCR Master Mix using the following oligonucleotide primers: NF-t-test by SPSS 17.0 software. Differences were considered significant when P < 0.05.Datas are presented as mean ± SD. Statistical analysis was performed with the independent-samples On the third day of tail intravenous injection of ADR, proteinuria in the rats was increased gradually. Rats in model group presented lower food intake and less activity compared with those in control group. However, rats in treatment groups showed a better mental condition, raised movement and food intake, and a better hair sheen.P < 0.01)  .P < 0.01) but decreased levels of serum TP and ALB (P < 0.05). These phenotypes were similar to the clinical symptoms of nephrotic syndrome. Furthermore, ZWT at the dosages of 24.0 g/kg/d and 12.0 g/kg/d for 28 days significantly prevented the increase in serum CHOL, TRIG, Scr, and BUN and the decrease in serum TP and ALB.As depicted in Long-term NS may induce changes in the conditions of kidney. In the present study, kidney of ADR-treated rats showed severe edema and less luster compared with the control group. The kidney in ZWT treated group (24.0 g/kg/d and 12.0 g/kg/d) presented more dense shape and redder look . MoreoveIn our study, we found that treatment with ZWT at the dosages of 24.0 g/kg/d and 12.0 g/kg/d for 28 days could significantly prevent the increase of renal IL-8 and the decrease of renal IL-4 induced by ADR Figures . ZWT couP < 0.01). Further, treatment with ZWT decreased the strong fluorescence induced by ADR (P < 0.01)  .Renal pathological examination is a fast, clear, and direct method in diagnosis of nephrotic syndrome. In model group, glomerular extracellular matrix (ECM) accumulation and glomerular mesangial cell (GMC) proliferation and base-membrane thickness were observed. Further, foot processes were wide effacement and the width of foot process was much larger than that of normal control rats . Under tκB plays a key role in the regulation of cytokine expressions. Because most cytokines levels in NS are partly or predominantly regulated by NF-κB, we postulated possible involvement of NF-κB in NS. In the study, the results showed that the mRNA expression of NF-κB p65 in the renal tissue of ADR-induced NS rats was highly upregulated and that of IκB was highly downregulated compared with the control group. However, ZWT could decrease the expression of renal NF-κB p65 but increase the expression of IκB (P < 0.01) (NF- < 0.01) .ZWT is a blended traditional Chinese medicine specifically for the treatment of various renal diseases. The present study demonstrated that ZWT improved ADR-induced nephrotic syndrome. NS is a series of clinical symptoms, including proteinuria, hypoalbuminemia, edema, and hyperlipidemia . ProteinThe renal function is closely associated with urine protein excretion level and a direct reflection of disease's progression . The conDyslipidemia is also one of the main phenotypes in ADR-induced NS in the rats. Consistent with previous reports , our pre+ Th2 lymphocytes. IL-4 is the primary cytokine for the spontaneous production of IgE and IgG by peripheral blood mononuclear cells, which is associated with immune complex depositing at glomerular basement membrane. However, its role in NS is controversial. In some researches, it was reported that IL-4 was increased in NS rats [α (TNF-α) production by macrophages and upregulation of anti-inflammatory cytokines such as TGF-β [A possible role for Th2 cytokines in the induction of proteinuria in minimal change nephropathy (MCN) is supported by clinical observations, such as the association of MCN with atopy and the apparent induction of MCN by allergic events in some patients , 28. IL- NS rats , and IL- NS rats . In some NS rats . In the as TGF-β , 33. We as TGF-β . In the as TGF-β , 36. Furas TGF-β . ImmunofκB is a transcription factor activated by cell surface receptor signaling to meet stress and inflammatory responses, regulating key cellular processes such as inflammation, innate and adaptive immunity, and cell growth and survival [κB family of transcription factors has been shown to regulate various aspects of T-cell, including Th1, Th2, Th17, Th9, and Tfh cells [κB is sequestered in the cytoplasm bound to IκB; phosphorylation of IκB releases active NF-κB, which translocates to the nucleus to induce an extensive range of target genes [κB activation and ADR induced NS and whether ZWT might regulate this pathway. To test this, we examined NF-κB (p65) and IκB mRNA expression in kidneys of a rat NS model induced by ADR. In our experiment, NF-κB (p65) mRNA expression in model group was significantly upregulated, but IκB mRNA expression was significantly downregulated compared with control group. And the results are consistent with what has been reported. The results suggest that the activation of NF-κB pathway was involved in the pathogenesis of NS. ZWT could inhibit the activation of NF-κB and prevent kidneys from being injured.NF-survival . Notablyfh cells . It is cfh cells , 40. In et genes . In respAntioxidant therapy is another strategy for NS treatment and is considered to be an important pathogenic mechanism. Oxidative stress develops from an imbalance between oxygen free raddical (ORF) production and reduced antioxidant defenses, such as SOD, CAT, glutathione, and glutathione peroxidase , 43. ORFTaken together, we propose that ZWT could ameliorate the proteinuria, low serum albumin, hypercholesterolemia, and loss of kidney function of NS rats. These data provide direct evidence for ZWT-treated nephrotic syndrome, at least in part, by modulating the balance between inflammatory and anti-inflammatory responses, enhancing antioxidant capacity and the elimination capacity of ROS. However, the active compounds in ZWT responsible for its treatment are not revealed at present, and its more protection mechanisms are also not clear. Therefore, we will continue to investigate further. Network pharmacology and membrane immobilized chromatography would be involved in our follow-up study.


A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during  In vitro maturation (IVM) of mammalian oocytes is an important technique for animal embryo technology, which directly affects the efficiencies of in vitro fertilization (IVF), nuclear transfer and transgenic etc. The developmental competence of oocytes matured in vitro is generally lower than those matured in vivo1in vitro, we must understand the molecular mechanism of oocytes during maturation, determine key genes and proteins associated with oocyte maturation, and then find a reliable molecular marker to predict developmental competence of oocytes matured in vitro.During mammalian oogenesis, the oocyte undergoes a protracted period arrest at dictyate or germinal vesicle (GV) stage, subsequently undergoes germinal vesicle breakdown (GVBD) and proceed to the metaphase I (MI) stage, followed by extrusion of the first polar body and arrest at metaphase II (MII) stage . Only deProteomics have been applied in the research of mammalian oocytes and embryos, including mouse891011131461617Isobaric tags for relative and absolute quantitation (iTRAQ) is a widely used stable isotope-based approach for quantitative proteomics, which allows simultaneous identification and quantification. The same peptide from different samples display a single peak in MS scans, thus reducing the complexity of parent ion spectra. And quantification is performed via reporter ion intensity from the low mass range at MS/MS level, which improves the accuracy of quantitation. In addition, this method could simultaneously analyze up to eight different samples in one experiment.Bubalus bubalis) is an important domestic animal distributed in the tropical and subtropical region, providing high quality of milk, meat, and work powerin vitro is reported to be low in comparison with bovine blastocysts produced in vitro. Thus, the present study was undertaken to investigate the protein expression profile of buffalo oocytes during IVM, identify the differentially proteins in oocytes at GV and MII stages with different competence using iTRAQ quantitative proteomics technology. This research will provide useful information for understanding the changes in protein profiling of buffalo oocyte during maturation, and then set up a foundations for further exploring the molecular mechanism of buffalo oocytes matured in vitro.Buffalo  and embryos . Proteome differences in the bovine and buffalo oocytes were showed as Venn diagram of absolute protein numbers  compared to GV stage oocyte (GVO). Among these differentially expressed proteins, 108 and 65 proteins were up-regulated and down-regulated respectively. When MII G were compared with incompetent MII oocytes (MII B), 146 differentially expressed proteins (111 up- and 35 down-regulated) were found to be mapped the cut-off criteria , Fig. 5.To understand the dynamic changes of proteins expressed differentially during buffalo oocyte maturation, hierarchical cluster was performed. Proteins clustered were those differentially expressed at least in one of the two pairwise comparisons. As showed in in vitro maturation of buffalo oocytes.To further reveal the signaling pathways related to the maturation of buffalo oocytes, proteins related KEGG pathway were analyzed. As shown in in vitro maturation, quantitative RT-PCR was performed to check expression of five genes . As shown in To further demonstrate the proteins expressed differentially in buffalo oocytes during In vitro maturation of oocytes is an important technology for providing matured oocytes that are utilized in IVF, in vitro production (IVP), and somatic cell nuclear transfer. In the past decade, although many efforts have been made to improve the efficiency of the IVM, the development competence of oocytes matured in vitro is lower than oocytes matured in vivo1in vitro. Proteomic approaches allow us to monitor dynamic changes at protein expression level and identify proteins that are functionally associated with a special cell or tissue phenotype. Knowledge of protein expression profiles occurred during the process of oocyte maturation will provide new insights into the molecular mechanisms regulating oocyte maturation.in vitro maturation and a total of 3,763 proteins were identified, which represented the largest buffalo oocyte proteome dataset so far. The identified proteins were known to be essential for oogenesis and embryo development, which previously detected in other species, such as NLRP5/MATER, OOEP/FLOPED, PADI6, PRDX1, GDF9, NPM2, TLE6. Novel proteins were also identified in buffalo oocytes, such as ZAR1, BMP15, DNMT1, and PTPN1 , fatty acid metabolic, lipid biosynthetic and steroid metabolic. These results indicated that oocytes may require different metabolites  to support their growth and maturation. During the maturation process, oocytes synthesized and stored a large amounts of mRNA and proteins in the cytoplasm, and this materials were utilized later in embryo development until the embryonic genome was activated21in vivo-derived oocytes was significantly higher than that of poor or matured in vitro oocytes2727The important purpose of this work was to investigate the protein expression difference in buffalo oocytes with different maturity or quality. A total of 173 proteins differentially expressed was identified in immature and mature oocytes, and 146 proteins differentially expressed were identified between competent and incompetent matured oocytes. Among these differentially expressed proteins, proteins involved in the oxidative phosphorylation pathway and enriched in mitochondria were up-regulated in MII G compared to GVO and MII B oocytes. Oxidative phosphorylation (OXPHOS), one main pathway occurred in mitochondrial, is the important physiology process to generate ATP24In the present study, a large group of proteins associated with protein transport and transmembrane transport were also found to be up-regulated in MII G oocytes compared to GVO and MII B oocytes, indicating that substrates transport were necessary for the buffalo oocyte meiotic maturation. RAB family proteins included RAB2A, RAB3A and RAB21, which were associated with signal transduction, intracellular vesicular transportThree major classes of molecular motor are kinesins, dyneins and myosin4543Ribosome is ribonucleoprotein complexes comprising RNA and protein, whose major function is responsible for protein synthesis. The protein synthesis is essential for oocyte meiotic maturation and subsequently embryo developmentet al. demonstrated that inhibition of UPP prevented cyclin B1 degradation, inhibited PB2 extrusion and pronuclear formationFurthermore, several proteins  related to ubiquitin-proteasome proteolytic pathway were found to be up-regulated in MII G oocytes in the present study. Ubiquitin-proteasome proteolytic pathway (UPP) is the main routes for intracellular protein degradation in eukaryotic cellsIn conclusions, the expression level of proteins in buffalo oocytes is related to their physiological states, and the up-regulated proteins in competent MII oocytes compared to GV and incompetent MII oocytes are related to chromosome segregation, microtubule-based process, protein transport, ribosome, UPP, and OXPHOX. The oxidative phosphorylation activity may be important for the meiotic resumption and competent acquisition of buffalo oocytes during maturation.4HCO3) were obtained from Amresco . Protease inhibitor complete tablets was purchased from Roche. iTRAQ-4plex Regent kit was obtained from Applied Biosystems . Trypsin was from Promega . All the water was prepared using a MilliQ system .Tissue culture medium 199 (TCM 199) and fetal calf serum were purchased from Gibco BRL . Follicle-stimulating hormone, formic acid (FA), bis-acrylamide and acetonitrile (ACN) were supplied by Sigma . Urea, iodoacet-amide (IAA), ammonium bicarbonate (NHin vitro maturation (IVM). Then, COCs were cultured in droplets of TCM 199 medium supplemented with 10% fetal calf serum and antibiotics at 38.5 °C in an atmosphere of 5% CO2 for 22–24 h. The GV and M II oocytes of which cumulus cells were removed by vortexing and pipetting were washed three times in PBS buffer and stored at −80 °C until use. According to the morphological evaluation, Oocytes were divided into three kinds, I: immature oocytes with intact germinal vesicle and multilayered, compacted cumulus (GVO); II: competent matured oocytes with homogenous cytoplasm, at least three cumulus layers, and the first polar body (MII G); III: incompetent matured oocytes with heterogeneous cytoplasm, incompact and heterogeneously pigmented, and surrounding with few cumulus cells (MII B).Buffalo ovaries were collected from the local slaughter house and transported to the laboratory in physiological saline at 25 °C. After washing in saline solution, cumulus-oocyte complexes (COCs) were aspirated from 2 to 6 mm follicles of ovaries and COCs with compact cumulus cell layers were selected for http://rsb.info.nih.gov/nihimage/).All of the GV, competent and incompetent MII oocytes were lysed in 20 μL of lysis buffer  protease inhibitor cocktail). The samples were vortexed for 1 min and incubated on ice for 30 s with a total of 15 cycles. The lysates were centrifuged at 13,800× g for 3 min and the supernatants were collected. After adding of SDS buffer, the proteins of oocyte lysates (20 μg of each sample) were separated by 10% SDS-PAGE, stained with Coomassie brilliant blue. The gels were scanned with a Scanjet image system (HP Scanjet G4050), the gel image was analyzed by Scion Image . Then, iTRAQ regents were dissolved in 70 μL of ethanol by vortexing for 1 min. Oocytes at GV stage were labeled with iTRAQ tags 114 and 115. MII G and MII B samples were labeled with iTRAQ tags 116 and 117, respectively. Tubes were incubated for 2 h at room temperature. The reaction was stopped by adding 120 μL H20, 2% ACN, pH 10.0) and separated by hiph-pH reverse-phase LC column . The 60 min liner gradient was composed of 96% buffer A for 1 min; 4–19% buffer B  for 30 min; then 19–95% buffer B for 23 min; followed by 95% buffer B for 5 min. The eluted fractions were collected in every 1.5 min, and then pooled into 20 fractions based on the peak intensity. The collected fractions were then lyophilized and stored at −80 °C until MS analysis.The pooled peptides were resuspended in 80 μL of buffer A  and analyzed by LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific. San Jose. CA). Peptide mixtures were injected into the capillary column (75 μm × 15 cm) and separated by a 3 μm C18 column using NanoAcquity ultra performance liquid chromatography (UPLC) system . Peptides were eluted with a linear gradient of 8–40% buffer B (0.1% FA in ACN) at a flow rate of 0.3 μL/min for 60 min. The mass spectrometer was operated in positive ion mode (source voltage 2 KV) and data-dependent manner. The full MS scans were performed in the Orbitrap at the range of 400–1,800 m/z at a resolution of 30,000. For MS/MS scans, the 10 most abundant ions with multiple charge states were selected for higher energy collisional dissociation (HCD) fragmentation following one MS full scan. Isolation window was set as 2.0 m/z. The dynamic exclusion was 35 s, and normalized collision energy of 40% was applied.Bos taurus protein database . False discovery rate (FDR) was estimated by using target-decoy based strategy. The proteins and peptides were filtered with a FDR < 0.01. The enzyme parameter was limited to semi-tryptic peptides with a maximum miscleavage of 2. Peptides with at least six amino acids were accepted. For protein identification, at least one identified peptide was required. Peptides precursor mass tolerance was 20 ppm, and fragment mass tolerance was 0.1 Da. Variable modifications of oxidation (+15.9949 Da) of methionine was selected, and Carbamidomethylated cysteine (+57.0215 Da), iTRAQ 4-plex (K) and iTRAQ 4-plex (N-term) were set as fixed modification.The MS/MS spectra were obtained and searched using MaxQuant  against Uniprot Protein with at least one unique peptide was validated and selected for further quantitative analysis. Only the peptide-spectrum matched (PSM) with complete reporter ion was allowed and the reporter ion intensity was picked up using the central MGF files. The isotopic correction was applied to reporter ion intensities according to the correction matrix provided by the manufacturer (Applied Biosystems). Peptide intensities were calculated by averaging the intensity of all the high confident PSMs of the peptide. The ratio of a protein is computed as the geometric mean of corresponding unique peptides ratios belonging to the protein. Significance B approach was performed to determine the p-value. Protein ratios greater than or equal to 2 or less than 0.5  were considered to be differentially expressed proteins.2O. The relative expression levels of targeted genes were calculated using the 2-ΔΔCT method. Three replicates were carried out for each gene using different sets of oocytes. The primer sequences used for the qRT-PCR analysis were showed in Total RNA was extracted from five oocytes at different stages  using Cells-to-cDNA II Kit . The cDNAs were reverse-transcribed using Takara RNA PCR kit (Takara) according to manufacturer’s instruction. Quantitative real-time RT-PCR analysis was performed using ABI 7500 PRISM system . The reaction system (20 μL) was consisted of 1 μL cDNA, 10 μL FastStart Universal SYBR Green Master (ROX) Mix, 0.5 μL of up and down primer (10 nM) and 8.5 μL ddHhttp://david.abcc.ncifcrf.gov/). SPSS 17.0 was applied to evaluate the statistical significance of mean values. Probability values less than 0.05 was considered to be statistically significant.GProXHow to cite this article: Chen, L. et al. Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique. Sci. Rep.6, 31795; doi: 10.1038/srep31795 (2016).


Mercurialis annua outlive male plants. This led to the hypothesis that female plants of M. annua would be more tolerant to stress than male plants. This hypothesis was addressed in a comprehensive way, by comparing morphological, biochemical and metabolomics changes in female and male plants during their development and under salinity. There were practically no differences between the genders in vegetative development and physiological parameters. However, under salinity conditions, female plants produced significantly more new reproductive nodes. Gender-linked differences in peroxidase (POD) and glutathione transferases (GSTs) were involved in anti-oxidation, detoxification and developmental processes in M. annua. 1H NMR metabolite profiling of female and male M. annua plants showed that under salinity the activity of the TCA cycle increased. There was also an increase in betaine in both genders, which may be explainable by its osmo-compatible function under salinity. The concentration of ten metabolites changed in both genders, while ‘Female-only-response’ to salinity was detected for five metabolites. In conclusion, dimorphic responses of M. annua plant genders to stress may be attributed to female plants’ capacity to survive and complete the reproductive life cycle.The research presented stemmed from the observations that female plants of the annual dioecious  Plants, as sessile organisms, encounter environmental conditions that play a key role in their evolutionary strategies for survival. Extreme environmental conditions were conducive for development of adaptive tolerance mechanisms, including diverse reproductive systems. It was suggested that the evolution of dioecy, where female and male functions are born on separate individuals, enables better capacity to cope with limited resources in stressful environmental conditions . A compr2O2, a less harmful molecule that is detoxified by various peroxidases, of which APX is prominent [Many of the stresses faced by living organisms involve the formation of reactive oxygen species (ROS), which are innate to life under oxygenic atmosphere. ROS are formed by normal metabolism, but their formation is enhanced under abiotic and biotic stresses such as: drought, salinity, developmental and stress induced senescence, herbivores and pathogens ,4,5. Plarominent  and the rominent  constitu13C-isotopologue profiles  [Changes in plant metabolism reflect the appropriate adjustments needed to cope with various stresses and may hold a clue to the metabolic pathways most affected by stress ,13. Recer, 2007) . Metabolr, 2007) ,16,17.Mercurialis annua (Euphorbiaceae), used in this study is a common roadside herb native to the Mediterranean basin, which has spread into Europe, North America and Australia [n = 16) M. annua species is a strictly dioecious annual, while the polyploid species are not [M. annua plants are indistinguishable and can be identified by gender specific molecular markers [M. annua, female plants keep producing seeds until late senescence, while the male plants enter the senescence stage before the female plants as seen in nature  [Silene latifolia did not respond differently to low resources availability, although they did reaffirm that females allocate more resources needed to reproduction than do male plants [M. annua   found the plants . These ie plants ,42. In tM. annua , led us  species . Similare leaves . In Ginke plants . The effe plants  and pople plants ,30.M. annua, served as model system to test the phenotypic and metabolic response of female and male plants to salinity.In this research, the dimorphic response of annual dioecious, M. annua was operatively divided into five developmental stages, from the ‘young’ to the ‘late senescence’ stages (The growth of ’ stages . These s’ stages . (e) ’la’ stages . The dif’ stages . This in’ stages  or a lon’ stages .M. annua had a higher number of nodes as compared to male plants and their controls  higher than the male activity. Both activities peaked at 70 days and then dropped continuously till reaching similar values between 84 and 103 days. At ‘late senescence’, activities of both genders increased ; yet, these later activities were not statistically different. Exposure of ‘mature’ (84 days) female and male plants to salinity did not affect their leaf APX activities  activity in leaf of ‘young’ female ifferent . Exposurifferent .p < 0.05) and female activity increased, however, not significantly (Peroxidase (POD) activity was significantly higher in male plants at the ‘young’, ‘mature’ and ‘late senescence’ stages . Generalficantly .M. annua plants from the ‘young’ until the ‘late senescence’ stages  was assigned a value of 1.0  for a short time (48 h) was determined with model substrates. Whereas GST activity measured with the standard model substrates, CDNB and DCNB, did not significantly differ in salt treated and control plants, nor in plants of different gender (data not shown), the rates determined with the diphenylether herbicide, fluorodifen, as substrate were both sex and salt dependent (Glutathione S-transferase (GST) activity in ependent . The femependent . A similM. annua plants was significant (p < 0.05)  . ASC con < 0.05) . At the  < 0.05) . During  < 0.05) .In salt treated ‘mature’ plants (84 days), ASC leaf content decreased more in male than in female plants . As a result, ASC content in leaves of NaCl treated female plants was higher by 47% than in leaves of NaCl treated male plants .2O2 content of male and female leaves during development, except at the ‘young’ and ‘senescence’ stages  and was lower by 67% in male than female leaves , carbohydrates (4), TCA cycle compounds (6) and secondary metabolites (7) were identified in polar extracts of leaves and roots detached from ‘young’ (55 days) M. annua plants exposed to salinity for 3 days (Using r 3 days .p < 0.05), however, there was no significant difference between the total female and male plant metabolites concentration  higher in male plants  was determined in sucrose, acetyl CoA, betaine and aspartate in mature female and male plants, with the exception that DMA had increased only in female plants. The decreased concentrations of lactate, valine, citrate, succinate and malate, under salinity, were common to both sexes. The decrease in ferulate, fumarate, threonine, and proline, under these conditions, was however unique to female plants. It should be noted that salinity did not significantly affect the concentration of the other measured metabolites.The changes in specific metabolite concentrations under salinity treatment are shown in M. annua plants express dimorphic responses at the physiological, biochemical and metabolic levels, under stress and non-stress conditions. There were no significant differences, until early senescence, between female and male plants in the vegetative parameters, i.e., stem and root lengths, photosynthesis, transpiration and stomatal conductance. Differences, however, were distinct at senescence and under salinity  number of reproductive nodes characterized senescing female plants in comparison to male plants  Dumort. and Spinacia oleracea die earlier than female plants [Populus cathayana and Ginkgo biloba, male leaves were earlier senescent than female leaves [The underlying hypothesis of this study was that female and male salinity . In M. ae plants . Similare plants , suggeste plants . This wae leaves ,45.i.e., senescence and under salinity  [2O2 scavenging. Furthermore, the higher POD activity in male plants, throughout development   classifielopment , may be 2O2 detoxification capacity may be compensated by a concomitant increase in ASC concentration; accordingly, the slight increase that was observed in females, peaking at ‘late senescence’, added to their potential to detoxify hydrogen peroxide . The increase in betaine in both female and male plants may be explainable by its osmo-compatible function under salinity. However, the decrease in proline in female only and the fact that the concentration of both compounds was very low may suggest that they do not function as a compatible solute in M. annua or that the short term of exposure to salinity was not enough for significant concentration build-up of these compounds . The seeds were germinated, within 4 to 6 days, on top of saturated potting soil and covered by another 2 cm layer of dry soil mixed with Osmocote Plus . About 25% of the seeds germinated after 3 days with an approximate 1:1 ratio of male:female. It took approximately 4 to 6 weeks for the plants to begin flowering.Seeds of M. annua plants were compared along their development, from the ‘young’ (55 days) to ‘late senescence’ (122 days), with and without salt (NaCl).The morphological, physiological and anti-oxidant systems of female and male 2O2 and MDA.Establishing a reference time course scale of development of female and male plants was done by growing the plants in soil and harvesting three plants of each gender every two weeks before noon, beginning from ’young’ until the ‘late senescence’ stage. Fully developed leaves were collected, halved along the main vein of the leaf, for assay of the enzymes and substrates on the same leaf in each plant and stored separately at −80 °C for assays of the anti-oxidative enzymes, APX, CAT, POD and SOD and substrates, ASC, HM. annua gown in soil were uprooted at day 84 (‘mature stage’) from sowing and transferred to 16 L containers containing aerated-half-strength Hoagland solution, with four female and four male plants per container. Two of the containers were used as control without NaCl and two for salinity treatment challenged with 100 mM NaCl. Three days after transfer to the hydroponic system, salt treatment began by the addition of 33.3 mM salt, with subsequent 33.3 mM daily incremental increases up to 100 mM NaCl. After eleven days in 100 mM NaCl, the leaves from the top 5th node were harvested, cut in half along the main vein, and stored at −80 °C for analysis. All samples were assayed for the anti-oxidative enzymes, APX, CAT, POD and SOD and substrates, ASC, H2O2 and MDA. In the case of the GST activity ‘early senescence’ plants (98 days) were exposed to NaCl (150 mM) for 2 days.The effect of salinity on female and male plants was conducted using ‘mature’ (84 days) plants grown in salinity for 11 days as follows: 32 plants  of 2 concentration of 380 μmol·mol−1, 25 °C and a photosynthetic photon flux density of 250 μmol·m−2·s−1.Photosynthetic, transpiration rates and stomatal conductance were measured in control and NaCl (100 mM) treated plants. In the experiment of the long term effect of salinity, plants were grown under salinity from one week after germination until the gas exchange measurements were conducted at days 42 and 59 after sowing. In the short term salinity effect experiment, 56 days old plants grown in soil without salt (control) were transferred to the salinity treatment (NaCl 100 mM) and the gas exchange measurements were conducted after 2 h and 3 days. In both experiments, 4 plants (of each gender) were analyzed in control  and 8 plants in salinity treatments. For each sampling time, five measurements were made on each plant. Measurements were done using a portable gas-exchange measuring system , at a COet al. (2000) [et al. (2005) [Total soluble proteins for anti-oxidative enzyme assays were extracted by grinding 1–2 g of plant material in a mortar and pestle in the presence of liquid nitrogen. Extraction was conducted according to Mittova . (2000) . The ext. (2005) . Protein. (2005)  with bovet al. (1997) [Ascorbate peroxidase  activity was determined immediately after the extraction by monitoring the decrease in optical density at 290 nm, according to Jiménez . (1997) .2O2 consumption [Catalase  activity was followed spectrophotometrically by the decrease in absorption at 240 nm, resulting from Hsumption .Superoxide dismutase  activity was determined by monitoring the inhibition of photochemical reduction of nitro-blue-tetrazolium (NBT) at 560 nm, according to Beyer and Fridovich (1987) . One uniGuaiacol peroxidase  activity was followed spectrophotometrically by the increase of absorption at 470 nm, resulting from tetra-guaiacol formation by enzymatic oxidation of guaiacol .et al. (2005) [−1·cm−1), 1, 2- dichloro-4-nitrobenzene , 4-nitrobenzyl chloride  and p-nitrobenzoyl chloride , respectively, as substrates. Assays using the herbicide fluorodifen (ε400 nm = 3.1 mM−1·cm−1) as a substrate followed Scalla and Roulet (2002) [Glutathione S-transferase  activity was assayed spectrophotometrically by the method of Schröder . (2005)  using 1-t (2002) .3+ to Fe2+ by ASC and was monitored at 525 nm [Reduced ascorbate content was measured spectrophotometrically via the reduction of Fet 525 nm .Lipid peroxidation was estimated by assessment of malondialdehyde (MDA) using the method of Draper & Hadley (1990) .Hydrogen peroxide content was assayed according to Wolff (1994) .M. annua at ‘young stage’ (55 days) were exposed to NaCl (100 mM) for 3 days. Extraction of polar metabolites from female and maleleaf and root was done with perchloric acid, based on the protocol by Bouzier et al. (2000) [2O. The solution was centrifuged. To the supernatant (550 μL), 3-(trimethylsilyl)-1-propanesulfonic acid was added to a final concentration of 0.19 mM as internal standard. 1H NMR was recorded at 500 MHz using a Bruker DRX 500 instrument equipped with an inverse 1H/13C probehead . NMR signals were tentatively assigned using Chenomx NMR suite 4.6.Plants of . (2000) . In brievs. male plants and organs (leaf vs. root) during development were conducted by Student T-test. One Way Anova—Fisher’s test was used in order to compare the specific activity of enzymes, metabolites and/or groups of metabolites in relation to gender, leaf, root and salinity. In the experiments involving enzymes, substrates and metabolites the data represents averages of at least 3 plants (indicated by n = 3 in the legends to figures and tables), each analyzed in triplicates.Statistical analysis was done using SigmaPlot 2000 Windows Version 6.10. Significance analysis comparing responses of female