Osteoporosis is a disease characterized by low bone mass and progressive destruction of bone microstructure, resulting in increased the risk of fracture. Previous studies have demonstrated the effect of naringin (NG) or treadmill exercise (EX) on osteoporosis, however, reports about effects of NG plus EX on osteoporosis are limited. This study was designed to investigate the impact of combined treatment with naringin and treadmill exercise on osteoporosis in ovariectomized (OVX) rats. Three months after bilateral ovariectomy, Seventy-five rats were randomly assigned to the following treatment groups: OVX, sham-operated (SHAM), NG, EX, or NG plus EX treatment. Treatments were administered for 60 days. Bone metabolism, bone mineral density, trabecular bone parameters, immunohistochemistry, and the bone strength were evaluated. Compared to the OVX groups, all treatments increased bone volume (BV/TV), trabecula number (Tb.N), trabecula thickness (Tb.Th), bone mineral density (BMD), and mechanical strength. NG + EX showed the strongest effects on BV/TV, Tb.Th, and biomechanical strength. Additionally, decreased C-terminal telopeptides of type I collagen (CTX-1) and enhanced osteocalcin (OCN) expression were observed in the NG + EX group. The present study demonstrates that the NG + EX may have a therapeutic advantage over each monotherapy for the treatment of osteoporosis. Osteoporosis is a common disease worldwide, and characterized by decreased bone mineral density and progressive destruction of bone microstructure, resulting in an increased bone fragility and risk of fracture4Naringin is a polymethoxylated flavonoid, the main active compound of traditional Chinese medicine Rhizoma drynariae, and possesses a variety of biological and pharmacological effects. Naringin acts as anti-inflammatory, anti-mutagenic, analgesic and antioxidant676ALPNO)2 (PGE2)Similarly, exercise is considered to have positive influence on bone morphology and the mechanical loading generated by physical activity has been speculated to aid in preservation and improvement of bone mass and strengthThere are few published studies investigating the effects of the combination therapy - physical exercise and drugs - for treatment of osteoporosis. The purpose of this study was to evaluate the effect of naringin combined with treadmill exercise on osteoporosis in ovariectomized (OVX) rats. After sixty days of treatment, the systemic bone metabolism condition and bilateral femur were evaluated by serum analysis, histology, histological and fluorescent analysis, immunohistochemistry, dual energy X-ray absorptiometry (DEXA), biomechanics, and micro-computed tomography (micro-CT) assessment. These measurements were performed to investigate the effects of different treatments on osteoporosis in OVX rats.All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Tianjin Hospital Ethics Committee of China. The methods were carried out in accordance with the approved guidelines.Healthy 3-month-old Female Sprague-Dawley  was provided by the Experimental Animal Center of the Tianjin Hospital. The experimental animals received humane care. The animals were housed in an air-conditioned environment (22 ± 2 °C), with a 12-h light/dark cycle (7:00–19:00) and were allowed free access to food pellets and water throughout the experiment.2O only. In order to obtain dynamic parameters of callus formation and remodeling, tetracycline (25 mg/kg) were injected intramuscular at 10 and 3 days before sacrifice. On the last day of treatment, blood was collected from the heart under ketamine anesthesia. After centrifugation, serum was obtained and kept at −80 °C until further analysis. Femur were dissected and 5 right femoral in each group fixed with 75% ethanol stored at 4 °C for undecalcification and 5 right femoral in each group fixed with 4% paraformaldehyde, the remaining femur with phosphate buffered saline stored at −20 °C until analysis of BMD, Micro-CT and femoral mechanical test.Seventy-five rats were randomly divided into five groups (15/group), and four groups were removal of the bilateral ovaries (OVX). The rats in the sham group were incision and sutured without removal of the ovaries. Three months after post-surgery, the four OVX group treated with vehicle, naringin (NG), treadmill exercise (EX) ,and naringin plus treadmill exercise (NG + EX) for 60 days. The sham group was orally treated with Hin vivo study, naringin was dissolved in 0.9% saline and concentrations of 300 mg/ml were prepared. NG and NG + EX group rats were gavaged once per day with 300 mg/kg naringin which was delivered in 0.9% saline. The OVX and sham group were orally treated with the same amount H2O only.Naringin was purchased from Xi’an Guanyu Bio-tech Company . The purity was over 98%. For the Rats allocated to EX and NG + EX group were exercised on a motorized treadmill five consecutive days/week at a 5% incline for 60 days. Rats ran in individual running compartments that contained an electronic grid attached at the base of each compartment. Rats were introduced to the treadmill-training protocol over a 1-week period, during which the speed was gradually increased from 10 to 20 m/min, and the duration of each session increased from 10 to 60 min/session. For the remaining 53 days, rats ran at 22–24 m/min for 60 min/session3 .The volume of interest (VOI) was selected as a region twenty-five slices away from the distal femur growth plate to 125 slices. The 3D images were obtained for visualization and display. Bone morphometric parameters including bone volume over total volume (BV/TV), trabecula number (Tb.N) and trabecula thickness (Tb.Th) were obtained by analyzing the VOI. The operator conducting the scan analysis was blinded to the treatments associated with the specimen.The BMD of right femur in each group (5/group) were measured by dual-energy X-ray absorptiometry (DEXA) . After DEXA measurement, the trabecular bone microarchitecture of the distal right femoral metaphysis was measured using a microtomography scanner , with a slice thickness of 21 μm and the voxel resolution of 22 μmAfter fixation in 75% alcohol for 7 days, the right femur (5/group) specimens were dehydrated in graded ethanol and embedded in methylmethacrylate  without decalcification. Seven μm-thick sections were obtained along the sagittal plane of the distal femur using tungsten carbide blades . Finally, sections were stained in giemsa for observation. The parameters were analyzed by Image-Pro Plus 6.0 software.The mechanical strength of the shaft and neck of the left femur (10/group) was measured with the method described by Ma and FuOsteocalcin (OCN) is a small noncollagenous matrix protein of the bone that derives from new cellular synthesis and a specific marker of bone formation whenever formation and resorption are uncoupled. Increased serum OCN reflects the acceleration of osteoblastic activity and bone turnoverFor immunohistochemical evaluation of OCN and CTX-1 expression (5/group), specimens were fixed in 4% buffered formaldehyde for 2 days at room temperature, and then decalcified in ethylenediaminetetraacetic acid (changed every 3 days) for 4 weeks. Decalcified tissues were then washed, dehydrated in gradient alcohol, embedded in paraffin wax, and cut into 4-μm-thick sections along the sagittal plane of the distal femur. Immunohistochemical localization of OCN and CTX-1 was carried out using commercially available antibodies according to the manufacturer’s suggested protocol . Negative controls were obtained by omitting the primary antibody. Stained sections were examined qualitatively under light microscopy  with a digital camera.Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using the statistics package SPSS16.0 . Multiple comparisons between groups were carried out using one-way ANOVA and Tukey’s post hoc test. P-values < 0.05 were considered statistically significant.All the rats recovered well after the surgery procedure. No animal deaths and weight loss were observed during the treatment period.p < 0.05). NG and NG + EX showed similar BMD of SHAM, but both were higher than that of EX (p < 0.05).Results of right femoral BMD by DEXA were presented in p < 0.01) lower trabecular BV/TV, Tb.N and Tb.Th, as compared to the sham rats. NG and NG + EX showed increased BV/TV than the VOX and EX (p < 0.05), while no difference in Tb.Th was observed between treated groups (p > 0.05). Moreover, NG + EX treatment presented the strongest effect on Tb.N than EX . NG, EX, and NG + EX showed similar effect on MAR, which was increased by 25.9%, 23.2%, and 28.7% compared to the VOX, respectively (p < 0.05). NG + EX showed the strongest effect on osteoclast perimeter (p < 0.05). NG and NG + EX showed similar osteoblast perimeter, while NG alone showed increased osteoblast perimeter and decreased osteoclast perimeter levels compared to the OVX group.In fluorescent analysis , NG + EXp < 0.05). Compared to the OVX group, NG, EX, and NG + EX groups increases of the average maximum fracture loading of left femoral neck were 20.4%, 13.1%, and 28.3%. NG, EX, and NG + EX group increases of the average maximum fracture loading of right femoral mid-shaft were 22.4%, 17.7%, and 29.3% ; while EX displayed similar OCN levels when compared to the VOX control (p > 0.05). For analyses of CTX-1 levels of NG, EX, and NG + EX decreased by 31.7%, 19.4%, and 36.1% compared to the VOX control (p < 0.05).Serum levels of OCN and CTX-1 were measured to provide an evaluation of bone formation and resorption activity under NG and/or EX treatment . NG and Immunohistochemical reactivity images for OCN and CTX-1 were exhibited in In the current study, we found that NG + EX exhibited greater therapeutic effects on osteoporosis than either monotherapy in OVX rats, with the highest values of BV/TV and Tb.N in micro-CT evaluation, significantly increased OCN and decreased CTX-1 levels identified in serum analysis, the strongest effects on fluorescence-marked rate and osteoclast perimeter in fluorescent analysis, and the strongest effects on femoral neck and femoral mid-shaft strength in biomechanical tests. These results indicated that the combined treatment of naringin and treadmill exercise had an additive effect on osteoporosis reduction in OVX rats.In this study, we used ovariectomized rats with the initial age of 3 months as a postmenopausal model because these rats are reproductively mature and capable of responding appropriately to estrogen deficiency and its sequela following ovariectomy. Three months were allowed to pass after surgery for the establishment of standard osteoporotic animal models as validated by literature23A three-point bending test and the femoral neck compression test was performed to evaluate the effect of NG + EX on the mechanical strength of the femoral shaft and femoral neck. The three-point bending test is a conducive test for cortical regions, and the femoral neck is the most important site for fractures in osteoporosisUndecalcified histologic sections are widely used to evaluate dynamic microstructural parameters of bone. Tetracycline is able to specifically bind calcium used in decalcification and thus be used to measure multiple parameters of bone tissue dynamics. According to the American Society for Bone and Mineral Research (ASBMR) proposed standard nomenclatureIn the present study, EX showed lower levels of osteoblast and osteoclast activity along the perimeter of bone in OVX rats. These findings are in agreement with the results of the previous studiesALP) activity of bone mesenchymal stem cells (BMSCs) in vitroin vitroLoss of bone mass was typically accompanied by a significant increase in bone remodeling, as was evidenced by altered levels of the bone turnover markers OCN and CTX-125et al. have demonstrated that estrogen receptors are located on the membrane surface of both osteoblasts and osteoclasts; however, estrogen functionality in bone resorption is inhibited and decreases the number of bone remodeling units34The mechanism of NG + EX is an interesting subject. The NG + EX treatment demonstrated the strongest effect on bone mass preservation and bone strength in OVX rats. Estrogen deficiency-induced osteoporosis belongs to a high conversion type in which bone formation and bone resorption are all increased, but bone resorption is more remarkablein vitro16in vivoIn addition, it has been confirmed that osteoblasts and osteoclasts sense and respond to mechanical stimuli29et al. demonstrated that the dose rate at 400 mg/kg of naringin by gavage daily for 2 months increased BMD of trabecular-rich bone in OVX mice. The dose rate at 200 mg/kg/day and 400 mg/kg/day of naringin showed similar increased stress-strain indices at the distal femur and lumbar spine and increased biomechanical strength at tibia diaphysis in OVX miceet al. reported that different concentrations of naringin  by gavage daily for 2 months treatmentet al. tested different intensities on the treadmill with ovariectomized adult rats. Moderate exercise was more efficient to increase BMD and bone strength of the tibia of the animals, while high-intensity exercise accelerated bone loss. Our training protocol was based on previous reports demonstrating that treadmill running is an osteogenic stimulus in ratsPrevious studies have reported doses of naringin  treatment on the retinoic acid-induced osteoporosis rats for 14 days. The results showed that the naringin-treated rats were superior to the untreated rats in the bone weight index, the length and the diameter of the femur bone, the bone ash content, and the levels of calcium and phosphorus. The dose of 100 mg/kg of naringin demonstrated greater effects on BMD of the femurThe mechanism of postmenopausal osteoporosis affecting bone impairment has not been completely identified. Although NG + EX in this study produced stronger effects than either monotherapy on osteoporosis in OVX rats within 60 days, longer observation time is needed for evaluation of the long-term efficiency of combined treatment. Furthermore, the physiological condition is different between OVX rats and postmenopausal women, and the treatment effects of naringin and exercise in this study is limited by the use of the animal model. The safety and efficacy of a combined intervention of naringin and exercise therapy should be further addressed in human models to determine the efficacy of this comprehensive treatment for post-menopausal osteoporosis.In conclusion, this study suggests that combined NG + EX held demonstrated greater effects on osteoporosis than either monotherapy in OVX rats. NG + EX produced the strongest effects on BMD, BV/TV, Tb.Th, MAR, and bone strength. NG + EX exerted locally increased OCN and decreased OCN levels in bone metabolism by serum tests. These results indicate that NG plus EX has an additive effect on osteoporosis associated with estrogen deficiency, and could potentially reduce the risk of fracture.How to cite this article: SUN, X. et al. The Effects of Combined Treatment with Naringin and Treadmill Exercise on Osteoporosis in Ovariectomized Rats. Sci. Rep.5, 13009; doi: 10.1038/srep13009 (2015).


Depression is the most widely acknowledged psychological problem among end-stage renal disease (ESRD) patients. Depression may be associated with VD deficiency. The aims of this study are to (a) elucidate the prospective association between HsCRP, VD contents and depressive symptoms in the dialyzed population, and (b) find the effect of calcitriol supplementation on depression in dialyzed patients.In this prospective study, 484 dialysis patients  from two hospitals in southeast China were included. The depression in these patients was evaluated using the Chinese version of Beck’s Depression Inventory (BDI). All subjects answered the BDI-I questionnaire for assessment of depression levels in summer. A cut-off value of 16 was set to include dialysis patients with depression. All patients were divided into two groups depending on the absence (Group1) or presence (Group 2) of depression. The two groups took 0.5 μg/day 1,25-Dihydroxyvitamin D orally for one year. BDI Scores were recalculated for all patients. Sociodemographic, clinical data, and serum VD contents were also collected.A total of 484 participants (247 men [51.0%] and 237 women [49.0%]) were surveyed. Depressive symptoms were found in 213 (44.0%) patients. The baseline serum VD level (VD2 + VD3) was 17.6 ± 7.7 nmol/L. Patients with depressive symptoms have significantly higher serum HsCRP level and significantly lower serum VD level compared with the control group. After one-year follow-up, the supplementation of 0.5 μg/day calcitriol slightly improved the microinflammatory state such as lowering mean serum HsCRP level and improving serum VD level, but not in significantly enhancing the depressive symptoms.Calcitriol supplementation did not significantly enhance the depressive symptoms in our dialyzed population although patients with low levels of serum VD were more depressed. Therefore, more prospective randomized controlled trials are necessary to reveal the exact cause-and-effect relationship between VD status and depressive symptoms or VD status related to some specific subtypes in dialyzed patients. Depression ranks fourth on the World Health Organization (WHO) global disease burden list. Depression is the most widely acknowledged psychological problem among end-stage renal disease (ESRD) patients ,2. AboutVitamin D (VD) is an essential nutrient for bone health, and has other physiological functions. There are plausible reasons for investigating the curative effects of VD for depressive disorders. Depression may be associated with VD deficiency -12. HypoHowever, it is unknown whether serum VD content is associated with depression in the dialyzed population and whether calcitriol supplementation is efficient for treatment of depression. The intentions of this study were to (a) investigate the prospective association between HsCRP, VD contents and depressive symptoms in the dialyzed population, and (b) find the efficacy of calcitriol supplementation on depression in dialyzed patients.In this prospective study, we included a total of 484 patients receiving dialysis  from two hospitals in China. All patients aged 18–60 who had received HD or PD for more than 3 months were screened for participation. Signed informed consent was required for enrollment, and after that, patients received oral and written information about the study. Ethical permission for the study was obtained from the Scientific Ethical Committee in Beilun Branch of the First Affiliated Hospital of Zhejiang University .Exclusion criteria: 1, cognitive deficits such as considerable memory loss, confusion/dementia, and intellectual disability; illiteracy and/or incapability of answering the questionnaire ; 2, Evidence shows that the incidence of depression may increase in older patients compared with normal adults . Thus, tDepression is a condition characterized by depressed mood or loss of interest or pleasure in nearly all activities almost every day for at least 2 weeks. The presence of depressive symptoms was determined using a Chinese version of Beck’s Depression Inventory (BDI) in summer, and the Chinese version was previously validated as the English version -20. The All patients were divided into two groups depending on the absence or presence of depression. In China, dialysis patients usually took 1,25- Dihydroxyvitamin D  orally 0.25 - 0.5 μg/day or 1.25 μg - 2.5 μg twice a week. Therefore, the two groups took 0.5 μg/day calcitriol orally for one year, and BDI scores were recalculated.Demographic data including employment status and education status were collected by questioning the patients. Information concerning age, sex, primary kidney disease, dialysis modality, comorbidities, health insurance, and mean time on dialysis (months) was gathered from medical records. Comorbidity was scored on the number of comorbid conditions using the comorbidity index according to a previous study .Blood was collected according to a standard protocol. Laboratory data including levels of intact parathyroid hormone (iPTH), serum albumin, high sensitive C-reactive protein (hsCRP) and serum calcium were gathered from medical records. Serum VD (VD2+ VD3) levels were analyzed using liquid chromatography–mass spectrometry  at t25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum were detected with HPLC-MS according to a previous study . Human s2 test as appropriate. The effects of the VitD treatment were examined using 2x2 ANOVA.We calculated and proved that a sample of 484 patients provided adequate power (α = 0.05 in two-sided t-test) for the proposed tests in this two-group study. Changes in HsCRP, VD contents and BDI scores from the baseline to the one-year follow-up were presented in a figure with 2 panels of the mean and error bars of each variable. The results were expressed as mean ± standard deviation (SD) and P < 0.05 was considered significant. The prevalence of depression was evaluated using frequency analysis and 95% confidence intervals (CIs). Differences in means were tested using an independent t-test, or Mann–Whitney test. Quartiles were compared with one-way analysis of variance (ANOVA), Kruskal–Wallis test, or χThe independent risk factors associated with BDI scores in dialysis patients were defined using multiple linear regression analysis. Depression can be affected by many factors including sex, marital stage, employment status, health insurance, living, comorbidity index, dialysis modality, hemoglobin, serum albumin, sensitive C-reactive protein, and VD. Therefore, all analyses were adjusted for those variables. All statistical analyses were evaluated using SPSS 17.00 .The 484 respondents were aged 52.7 ± 15.9 years on average. Their general characteristics are presented in Table The baseline serum VD (VD2+ VD3) level of the dialyzed population was 11.9 ± 6.8 (3.6 to 32.5) nmol/L. Overall BDI score for the studied population was 17.3 ± 7.2 points. Moderate to severe depressive symptoms (BDI score > 16) were found in 213  patients. The depression group has significantly higher serum HsCRP level  and significantly lower serum VD level  compared with the non-depression group.The independent risk factors associated with BDI score in dialysis patients were defined using multiple linear regression analysis. The independent variables such as age, sex, marital stage, employment status, health insurance, living, comorbidity index, and dialysis modality were inserted in the analysis with the BDI scores as criterion. Only sex and comorbidity index were independently associated with BDI scores, indicating that (1) The females were more likely to have higher BDI scores compared with males; (2) the dialysis patients with more comorbid conditions were more likely to have higher BDI scores compared with those with less comorbid conditions; (3) Patients with higher BDI scores have significantly higher serum HsCRP level and significantly lower serum VD level, not vice-versa.Data on serum HsCRP level, VD (VD2+ VD3) levels, and BDI scores after one year follow-up are listed in Figure This prospective study provided a comprehensive and detailed description of the incidence of depression and the effect of calcitriol supplementation on treatment of depression in dialyzed patients. The overall incidence of depression was  in the dialyzed patients, which was consistent with previous reports -32. The The depressive patients have higher serum HsCRP level than the non- depressive patients, and thus CRP may be as a new risk factor of depression in the dialyzed population. Also HsCRP level can be used to predict the outcome in HD patients. After one-year follow up, calcitriol supplementation slightly improved the level of mean serum HsCRP, which was not accompanied by the improvement of depressive symptoms. The causality between depression and inflammation is not well clear. Depression was linked to worse nutritional status in ESRD patients,  indicatiWe did find an association between depressive symptoms and serum VD (VD2+ VD3) levels in the dialyzed population. The patients with higher serum VD levels at baseline tended to have fewer depressive symptoms. Recently, the possible link between depression and VD status has been investigated in several small studies, but the findings are inconsistent because of differences in races, study populations, geographic locations and methodologies -39. In pThe effect of VD in the brain is not well understood. VD is considered as a neurosteroid across the blood–brain barrier. The VD receptors widespread in the brain include the hippocampus, which is associated with the development of depression. In our intervention, we did not find a significant effect of calcitriol supplementation on enhancing the depressive symptoms in the dialyzed population, since the effects are also conflicting -43. TherA few limitations in the current study should be noted. Specifically, although we assessed a broad range of depressive disorders, it is difficult to ascertain the specific causes of depression within the sample. Second, We did not subdivide the patients with depression: one group treated with the active compound and one treated with placebo. It was not a randomized double-blind trial study. Thirdly, the dose of 0.5 μg calcitriol for dialyzed patients is far enough for curing depression. Moreover, the sample in our study is far smaller than enough.Even though people with low levels of serum VD were more depressed, Calcitriol supplementation did not significantly enhance the depressive symptoms in our dialysis population. More prospective randomized controlled trials are necessary to find the exact cause-and-effect relationship between VD status and depressive symptoms or VD status related to some specific subtypes in dialyzed patients.The authors report no conflicts of interest. The authors alone were responsible for the content and writing of the paper.JZ, BB and CH carried out the literature study, data collection, data analysis and drafted the manuscript. The presence of depressive symptoms among patients was determined using a Chinese version of Beck’s Depression Inventory (BDI) by MN and JD. PZ and JC critically revised the manuscript. XN and YW performed data analysis and critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/14/125/prepub


Motility parameters were evaluated by Computed Assisted System Analysis (CASA). Experiment 2 was designed to study a high (50 μM) and low dose (12.5 μM) of H2O2 compared to a control (0 μM). Samples were incubated and further used for in vitro fertilization. Analyses of motility (CASA), oxidative status (CellROX green and 2'-7' dichlorofluorescein diacetate), mitochondrial potential (JC-1), chromatin integrity (AO), and sperm capacitation status (chlortetracycline) were performed. Embryos were evaluated based on fast cleavage (30 h.p.i.), cleavage (D = 3), development (D = 5), and blastocyst rates (D = 8). We observed a dose-dependent deleterious effect of H2O2 on motility and increase on the percentages of positive cells for CellROX green, capacitated sperm, and AO. A decrease on cleavage and blastocyst rates was observed as H2O2 increased. Also, we detected a blockage on embryo development. We concluded that sperm when exposed to oxidative environment presents impaired motility traits, prooxidative status, and premature capacitation; such alterations resulting in embryo development fail.Our study aimed to assess the impact of sperm oxidative stress on embryo development by means of a dose-dependent model. In experiment 1, straws from five bulls were subjected to incubation with increasing H In vitro embryo production (IVP) in human represents an alternative for couples who are unable to naturally conceive, even after programmed intercourse or artificial insemination [ in vitro produced embryos worldwide [mination . On the orldwide . HoweverOne of the reasons for the inconsistent results of IVP is the individual effect of bull, known to strongly influence embryo development capacity , 4. ThisIn vitro and in vivo embryo production systems have some disparities with an important difference associated with oxygen concentrations. Values of approximately 20% of oxygen in the air normally used in IVP labs are superior to those found in the oviduct and uterus of most mammals [ mammals . The exp mammals . This re mammals . Also, i mammals  verified in vitro fertilization is frozen semen. The process of cell cryopreservation has been related to ROS overproduction leading to cellular damage, especially due to lipid peroxidation, in different species including bovine sperm [Another factor that may intensify sperm oxidative damage, influencing IVP results, is the process of cryopreservation, considering that the main source of male gametes for bovinene sperm –20. Alsone sperm . in vitro embryo development. We evaluated the effect of oxidative stress induced by hydrogen peroxide on bovine sperm attributes  and subsequent in vitro embryo development. Furthermore, we propose a new and more sensitive flow cytometry method (CellROX green) to assess oxidative status of bovine sperm prior fertilization.ROS generation in the spermatozoa can occur in the electron transport chain or though the NADPH oxidase activity . Sperm eThis study was carried at the Animal Reproduction Department from the School of Veterinary Medicine and Animal Science of the University of São Paulo (VRA/FMVZ/USP). All procedures were performed according to the Bioethics Committee of the previously mentioned institution (protocol number 2710/2012).All chemical reagents and solutions used in this study were purchased from Sigma-Aldrich  unless otherwise stated.n = 5), donated from Reproduction Centers, were used. Bulls were selected according to post-Percoll motility of at least 70%. This experiment was conducted in four replicates comprehended in a period of 2 weeks.For this first experiment, thawed straws of the same batch from five Nelore bulls ( in vitro fertilization (IVF), experiment 1 was conducted as follows: each straw (0.25 mL) was thawed at 37°C for 30 seconds and subjected to Percoll gradient (45% and 90%) at 9000 G/5 minutes. Motile cells (pellet) were recovered and washed with 1 mL of Sp-TALP [6 sptz/mL in Fert-TALP [2 in air, and high humidity.In order to maintain the same condition of semen processing prior to Sp-TALP  at 9000 ert-TALP , with noμM. Hydrogen peroxide is not a free radical ; however, this peroxide is considered an important reactive oxygen species due to the high capacity to move across biological membranes and the high affinity with iron and copper ions to produce more unstable and reactive radicals such as the hydroxyl. Experimental groups were 0 (control), 12.5, 25, and 50 μM of hydrogen peroxide.For oxidative stress induction, we used hydrogen peroxide 30%  diluted in Fert-TALP, for a final solution of 625 μm/s; progressive motility > 50 μm/s; straightness > 70%. In brief, each slide was heated at 37°C; 5 μL of sample was placed in the slide and covered by a coverslip. A minimum of six fields were selected for analysis. Motility related variables considered were VAP (velocity average path), VCL (curvilinear velocity), VSL (straight-line velocity), BCF (beat cross frequency), ALH , total and progressive motility, and percentage of cells with fast, medium, slow, and static movement. Among the previously mentioned variables, total and progressive motility were selected as more relevant for the selection of the hydrogen peroxide concentrations used in the second experiment.Motility parameters were evaluated using the Computer Assisted Sperm Analysis system . Settings used, previously described by Goovaerts et al. , were 30μM (high concentration) and 12.5 μM (low concentration) and a control group (0 μM). This induction aimed to compare the impact of oxidative stress on cleavage, embryo development, and blastocyst rates. This experiment was conducted in 10 replicates, during two months, using 200 to 220 oocytes per replicate. For IVP control, semen samples with no H2O2 and not submitted to incubation were used and we considered only replicates with control blastocyst rate ≥20% (data not shown).In this second experiment, for IVF, we used semen samples subjected to only two concentrations of hydrogen peroxide, selected according to experiment 1 results: 50  in vitro maturation (IVM). COCs selected were washed 3X in holding medium (TCM199 Hepes supplemented with 10% FCS (Gibco), 22 μg/mL pyruvate, and 50 μg/mL gentamycin) and 3X in IVM medium , 50 μg/mL human chorionic gonadotrophin , and 1 μg/mL of 17β-estradiol) and placed for maturation in 90 μL microdroplets of IVM medium (20–30 oocytes/drop), covered with mineral oil, during 22 to 24 hours at 38.5°C, 5% (v/v) CO2 in air, and high humidity.Ovaries obtained from a slaughterhouse were transported to the laboratory in saline solution 0.9% at 30°C. Cumulus-oocytes complexes (COCs) were aspirated with an 18-gauge needle from 2 to 8 mm follicles. Oocytes with homogeneous ooplasm surrounded by more than two layers of compacted cumulus cells were selected forμM). However, after concentration adjustment, we performed a pool of 3 bulls (same number of cells of each bull), in order to eliminate sire effect, and then divided the same sample between experimental groups. Matured oocytes were washed 3X in pre-IVF medium (TCM199 Hepes supplemented with 0.003% of BSA-V (m/v), 22 μg/mL pyruvate, and 50 μg/mL gentamycin) and 3X in Fert-TALP and placed for fertilization in 90 μL microdroplets of Fert-TALP (20–30 oocytes/drop) covered with mineral oil. At the end of semen incubation, samples were washed with 550 μL of Fert-TALP (9000 G/90 seconds). The sediment with sperm was recovered and used to inseminate microdroplets with oocytes (±100.000 sptz/drop) and the rest of the sample was used in subsequent sperm evaluations.For IVF, the same semen processing and induction protocol described in experiment 1 were performed , putative zygotes were mechanically denuded by pipetting in pre-IVF medium and cultured in KSOM medium  during 8 days at 38.5°C, 5% CO2, 5% O2, and 90% N2, under high humidity. On third day of culture (D = 3), KSOM was supplemented with FCS to a final drop concentration of 5%.After IVF  and flow cytometry evaluations . This latter equipment contains a blue laser, which operates at 488 nm and emits a 20 mW visible laser radiation. A total of 10,000 events per sample were analyzed and data corresponding to yellow , red , and green fluorescent signals  were recorded after a logarithmic amplification. For data analysis, cell doublets and debris were excluded using PM3/FSC (forward scatter) and all data was analyzed by FlowJo v10.2 software, except DNA integrity, which was evaluated using FlowJo v8.7 software.μM in DMSO), in the dark at 37°C. Samples were analyzed by flow cytometry after 10 minutes, excited at 488 nm, and detected at 590 nm. For positive control, we used the protocol described by Celeghini et al. [Mitochondrial membrane potential was evaluated by JC-1 probe  . This probe emits green or red-orange fluorescence for low (LMM) or high mitochondrial potential (HMP), respectively. The procedure was performed with 187,500 cells diluted in Fert-TALP and stained with JC-1  and 515–530 nm (DCFH). For data analysis, we selected the population of cells PI-DCFH+ .For this assay, 187,500 cells were stained with a solution containing DCFH and propidium iodide (PI) at a final concentration of 9.3 μM) for 30 minutes at 37°C, and, in the last 10 minutes, PI was added to a final concentration of 6 μM. Samples were analyzed by flow cytometry, excited at 488 nm, and detected at 630–650 nm (PI) and 515–530 nm (CellROX green). For data analysis, we selected the population of cells PI-VD+ . For CellROX green validation, we used increasing concentrations of hydrogen peroxide , and the same sample stained with CellROX green was also stained with DCFH, in order to compare the two techniques.CellROX green  is a fluorescent probe that penetrates the cell and, when oxidized by intracellular free radicals, binds to DNA, emitting a more intense green fluorescence. For this assay, 187,500 cells were stained with CellROX green , and each sample was analyzed by flow cytometry after 5 minutes of incubation at 37°C, excited at 488 nm, and detected at 630–650 nm (red) and 515–530 nm (green). For positive control, a sample was incubated with hydrochloric acid  and, for negative control, samples processed as described in experiment 1 were used.Chromatin stability assay was based on sperm chromatin structure assay . Samples were then fixed with 5 μL paraformaldehyde 4%. An aliquot of this suspension was placed in a glass slide, mixed with DABCO solution  and glycerol (1 : 9), covered with coverslip, and kept at −20°C, protected from light until the evaluation. Two hundred cells were examined, under epifluorescence microscope (Olympus IX80), using magnification of 1000x with mineral oil. Filters of 355 and 465 nm were used for excitation and emission, respectively. Three cellular categories were classified: noncapacitated (even distribution of yellow fluorescent over the head), capacitated (only acrosome region stained in yellow), and reacted (no yellow fluorescent in the head).Capacitation status was evaluated by CTC assay as described by Ward and Storey  with somD = 3). Development rate was performed at fifth day of culture (D = 5), and, at this moment, embryos were classified in three categories according to developmental stage: noncleaved (NC), 2–4 cells, or 8–16 cells. Finally, at day eight of culture (D = 8), blastocyst rate was assessed. All these evaluations were made in stereomicroscope  with 63x of magnification and the percentage of all rates was made over the total number of oocytes.Fast cleavage rate assessment was performed 30 hours after insemination (30 h.p.i), counting the number of structures that already showed fist cleavage at this point. Cleavage rate was assessed in the third day of culture (μM of hydrogen peroxide were pooled). Results were reported as untransformed means ± SEM. All statistical analyses were calculated with a significance level of 5%.Statistical analysis was performed using the software Statistical Analysis System 9.3 . Data were tested for residue normality and variance homogeneity. Variables that did not comply with these statistical premises were subjected to transformations. We used PROC GLM for polynomial regression model in both experiments, considering treatment as main effect. On experiment 2, Spearman correlations analysis was performed to verify the correlation between variables analyzed, using PROC CORR procedure. In this case, groups were analyzed separately as control or treated  are present in Supplementary Material available online at http://dx.doi.org/10.1155/2016/8213071 (Table S1).In the first experiment, we observed a negative effect of increasing concentrations of hydrogen peroxide for all motility related variables. This effect was evident for the velocity patterns revealed by the variables VAP, VSL, and VCL, which significantly decreased for all hydrogen peroxide concentrations. This decrease also occurred for BCF, however, in a more moderate pattern . Total aμM . SimilarμM . Media vμM concentrations of hydrogen peroxide. The criterion for such selection was a concentration that caused considerable oxidative damage but with acceptable motility for IVF .Based on the straight-line equation generated for total and progressive motility (Table S1), we chose, for experiment 2, 12.5 and 50 In this validation, our results show more sensibility of CellROX green to detect increasing concentrations of hydrogen peroxide when compared to DCFH . The samp = 0.13) nor oxidative status analyzed by DCFH (p = 0.09).The dose-dependent effect of oxidative stress on sperm motility occurred in the second experiment, similarly to the first experiment Figures . HoweverWe observed an increase in the percentage of cells with no membrane alteration and stressed PI-VD+;  and capap = 0.15). Figures For IVP, all parameters related to embryo development showed treatment effect, except for the fast cleavage rate  and late (development rate and blastocyst) embryo development.Correlation data are shown in Supplementary Material (Table S4). When sperm was treated with hydrogen peroxide, there was a positive correlation between the percentage of cells with high mitochondrial membrane potential and cells PI-VD+, and a negative correlation between the percentage of cells with high mitochondrial potential and movement pattern .For the oxidative status related variables, also in treated group, there was a negative correlation between stressed cells with no membrane alteration (PI-VD+ and PI-DCFH+) with movement pattern, and the percentage of cells PI-DCFH+ also correlates negatively with the total and progressive motility.Finally, in the treated group, cleavage rate correlates positively with total and progressive motility, and blastocyst rate correlates negatively with the percentage of cells PI-VD+ and positively with velocity pattern . in vitro fertilization. The severity of such effect is dependent on the intensity of the oxidative challenge. Interestingly, this impact can be observed not only on cleavage stage but also during the development to blastocyst. In addition, we verified the dose-dependent detrimental effect of oxidative stress on sperm motility, capacitation, and chromatin integrity. The regression model used in the preset work enables more inferences than studies which performed only mean comparisons. We can also consider the functioning of our treatment (hydrogen peroxide as oxidative stress promoter) over different biological sperm functions , inferring over the effect of other doses not used in the present study, once the model generates a straight-line equation. Based on such equation, we may calculate the ideal hydrogen peroxide concentration for different desired effects . Also, for the first time, we reported the use of a new fluorescent probe, more efficient and sensible, to evaluate oxidative status by flow cytometry on bovine spermatozoa.In our study, we verified that sperm submitted to an oxidative environment negatively influence embryo development when used forIn experiment 1, we proposed a model to evaluate the effect of induced oxidative stress on bovine sperm motility patterns. Even though we used low concentrations of hydrogen peroxide in a short incubation period when compared to those previously used –39, our Hydrogen peroxide promotes a negative effect on motility patterns, but this effect apparently is not related to alteration on mitochondrial membrane potential. In boar sperm, hydrogen peroxide induces similar effect; reduction on sperm motility is not accompanied by impaired mitochondrial membrane potential or ATP concentrations . These aThe positive correlation between the percentage of sperm showing high mitochondrial membrane potential and those with signs of stress and no membrane alteration (PI-VD+) found in the treated group suggests that, in stressful conditions, intact and metabolically active cells would exhibit greater potential to release prooxidative substances. Similar relationship between mitochondrial membrane potential and ROS has been demonstrated in other cell types , 43. StuμM) promote a time- and dose-dependent effect on tyrosine phosphorylation of some proteins related to capacitation, with opposite effect under high concentrations (5 mM) [An important biological function of ROS on sperm physiology occurs in the capacitation process . The acts (5 mM) . In our s (5 mM) , 47. In In our study, oxidative status was assessed by two fluorescent probes: 2′,7′-dichlorofluorescein diacetate (DCFH) and CellROX green. We observed differences in oxidative status detection between these two fluorescent probes. While, for DCFH, no dose-dependent effect for hydrogen peroxide was detected, for CellROX green, we could clearly observe an effect of increasing concentrations of hydrogen peroxide, indicating a difference of sensibility between these two probes to assess oxidative status.To our knowledge, there is no previous report regarding the use of CellROX green in bovine sperm. A recent study validated CellROX deep red to detect oxidative status in ovine sperm , but in Both DCFH and CellROX green are considered nonspecific to the type of free radical detected, although some studies still use DCFH as a specific probe to detect hydrogen peroxide production . In addiThe different sensibility of these two probes was observed in the validation for flow cytometry . When weIn our experiment, we verified that sperm, when exposed to an oxidative environment, induce a dose-dependent effect on embryo development, from the first cleavage until blastocyst stage. The diminished cleavage rate probably occurred due to the lower percentage of motile cells as the concentration of the induction agent increased, which would then lead to impaired fertilization rates. In fact, the correlations found between motility and cleavage rate would agree with this hypothesis. In the control group, no correlation was found between motility and cleavage rate probably because, in this situation, sperm motility would remain high due to the Percoll gradient. However, in the treated group, there was a positive correlation between these two variables, suggesting that only under oxidative stressful conditions (the presence of hydrogen peroxide) sperm motility would strongly influence cleavage rates.Embryo development rates evaluated on the fifth day of culture demonstrated that most embryos block the development at the 2–4 cells stage; at this moment, embryos should normally be at 8–16 cells stage. The influence of such developmental block would result in lower blastocyst rates. We can speculate two possible causes: (1) sperm that experienced an oxidative environment, after fertilization, may carry metabolites that would promote oocyte intracellular damage such as lipid peroxidation and antioxidant depletion, which would then impair initial embryo development (2–4 cells); (2) sperm DNA abnormalities induced by oxidative stress block embryo development even before embryo genome activation, leading to cellular division failure. The first hypothesis is very difficult to prove, once techniques currently available to assess zygotes' intracellular damage compromise further embryo development. Results of previous studies –31 give μM), hydrogen peroxide was capable of promoting a dose-dependent increase in the percentage of positive cells. Shaman and Ward [In our study, we verified the effect of oxidative stress increasing the percentage of cells with chromatin alteration (AO+). Chromatin alterations in bovine sperm are relatively rare when compared to other species , 53, 54.and Ward  describe HOX clusters, noncoding RNAs, and paternally expressed imprinted loci. This strategic localization of histones is considered important epigenetic markers of paternal DNA, indicating genes related to early embryo development that must be immediately activated after fertilization [The retained histones on sperm DNA are not accidental. In an interesting study, Hammoud et al.  identifilization , 58. Howlization , exposinEmbryo development fails due to spermatozoa exposed to oxidative stress which has been previously observed in primate . In thisWith the advances in transcriptome research, several studies have identified the presence of RNAs on spermatozoa –62. SomeWe concluded that an oxidative environment can significantly impair bovine sperm motility pattern, oxidative and capacitation status, and DNA integrity. These changes would then reflect negatively on embryonic development from cleavage to blastocyst stage. Also, our study validated a new method to evaluate oxidative stress, CellROX green, which is more sensitive and efficient when compared to another assay normally used for sperm. More studies focusing on the moments between first cleavage and embryonic genome activation should be conducted aiming to better understand the deleterious effect of oxidized sperm in this particular period.Supplementary material provides statistic data information of sperm evaluations (experiment 1 and 2) and embryo development analysis not shown in the article.


P < 0.001), a smaller peak eGFR AUC%_12M (P < 0.001), and a larger negative eGFR slope_12M (P < 0.001) were associated with a higher risk of renal end point. Two calculated formulas: initial eGFR AUC%_12M and eGFR slope_12M were the best predictors. Our results demonstrate that the greater eGFR variability by AUC% is associated with the higher risk of progression to RRT.Greater variability in renal function is associated with mortality in patients with chronic kidney disease (CKD). However, few studies have demonstrated the predictive value of renal function variability in relation to renal outcomes. This study investigates the predictive ability of different methods of determining estimated glomerular filtration rate (eGFR) variability for progression to renal replacement therapy (RRT) in CKD patients. This was a prospective observational study, which enrolled 1,862 CKD patients. The renal end point was defined as commencement of RRT. The variability in eGFR was measured by the area under the eGFR curve (AUC)%. A significant improvement in model prediction was based on the −2 log likelihood ratio statistic. During a median 28.7-month follow-up, there were 564 (30.3%) patients receiving RRT. In an adjusted Cox model, a smaller initial eGFR AUC%_12M ( Chronic kidney disease (CKD) is an increasing worldwide public health problem associated with increased morbidity and mortality , 2. GreaPlotting slopes of 1/creatinine or estimated glomerular filtration rate (eGFR) versus time has been used as a tool for outcome variable in clinical studies . eGFR sln = 413) or whose follow-up period was less than 12 months (n = 1048) were excluded. Besides, patients with CKD stages 1 and 2 (n = 152) were excluded. The final study population consisted of 1,862 CKD patients. Between February 2002 and September 2008, 3,475 patients who joined the ICKD (Integrated CKD care program) prospective observation study from two affiliated hospitals  of Kaohsiung Medical University were included and followed until May 2010. CKD was defined by using the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines, and the CKD stage was classified using each subject's baseline eGFR . Patient2 = 186 × Serum  creatinine−1.154 × Age−0.203 × 0.742  [Kidney function was quantified by using eGFR derived from the simplified Modification of Diet in Renal Disease (MDRD) Study equation. The equation was eGFR mL/min/1.73 m female) . Serum c female) .The renal end point was defined as commencement of RRT. In patients reaching renal end point, renal function data were censored at the start of RRT. The other patients were followed until May 2010. The commencement of dialysis was determined according to the regulations by the National Health Service for dialysis therapy based on laboratory data, nutrition status, and uremic symptoms and signs.2/year. At least three eGFR measurements were required to estimate eGFR slope. Faster renal function progression was reflected in a larger negative value of the slope. In addition, eGFR AUC% was used to estimate the variability in eGFR. The rate of renal function decline was assessed by the slope of eGFR, defined as the regression coefficient between eGFR and time in units of mL/min/1.73 mt-test for continuous variables with approximately normal distribution, or by Mann-Whitney U test for continuous variables with skewed distribution. Cox proportional hazards analyses were used to investigate the relationships between different methods of estimating eGFR variability and eGFR slope with renal end point. The adjusted covariates included age, sex, a history of diabetes, hypertension, and cardiovascular disease, systolic and diastolic blood pressure, BMI, albumin, fasting glucose, triglyceride, total cholesterol, hemoglobin, eGFR, total calcium, phosphorous, CaXP product, uric acid, urine protein-to-creatinine ratio, ACEI and/or ARB use, and acute kidney injury episode. A significant improvement in model prediction was based on the −2 log likelihood ratio statistic, which followed a difference in likelihood ratio. The P value was based on the incremental value compared with the basic model. Differences were considered significant if the P value was less than 0.05.Statistical analysis was performed using SPSS version 17.0  for Windows. Data are expressed as percentages, mean ± standard deviation, or median (25th–75th percentile) for triglyceride, urine protein-to-creatinine ratio, and days of follow-up. The number of all missing baseline data was less than 2% except the data of ACEI and/or ARB use. This adjustment of ACEI and/or ARB use was accomplished using multiple imputation approach. The differences between groups were checked by chi-square test for categorical variables, by independent A total of 1,862 nondialyzed CKD patients were included. The mean age was 63.6 ± 13.4 years and there were 1,084 males and 778 females. The underlying etiologies of CKD in our patients included 646 with diabetic kidney disease (34.7%), 647 with nondiabetic glomerular diseases (34.7%), 200 cases of hypertension (10.7%), and 369 caused by other diseases (19.8%). The comparison of baseline characteristics between patients with and without renal end point was shown in P < 0.001), small peak eGFR AUC%_12M , and negative eGFR slope_12M  were significantly associated with progression to renal end point. To clarify the effect of competitive risks between death and end-stage renal disease (ESRD), we further performed the multivariate analysis. We found that initial eGFR AUC%_12M , peak eGFR AUC%_12M , and eGFR slope_12M  were still significantly associated with progression to renal end point.The mean follow-up period was 28.7 ± 14.0 months. During the period of follow-up, five hundred and sixty-four patients (30.3%) who started RRT were recorded in these 1,862 patients. P < 0.001), peak eGFR AUC%_12M (P < 0.001), and eGFR slope_12M (P < 0.001) to the basic model significantly improved the predictive value of renal end points. The maximum change in the −2 log likelihood ratio was observed for initial eGFR AUC%_12M, followed by eGFR slope_12M, and peak eGFR AUC%_12M.The incremental values in eGFR variability and eGFR slope, when they were added to the basic model to predict renal end point, were shown in P = 0.590).Besides, with regard to using standard error (SE) of the regression as a method of valuating eGFR variability, we found that the SE of the regression was not significantly associated with renal outcomes in multivariate analysis . They used the coefficient of variation of the regression line coefficient fitted to all outpatient measures of eGFR to define variability in renal function and found that patients in the highest tertile of eGFR variability had an increased risk of death, independent of both baseline level of eGFR and eGFR slope. In addition, such variability provided further prognostic information beyond baseline eGFR and eGFR slope [The first important finding of our study is the identification of greater eGFR variability as a risk factor for adverse renal outcomes in CKD. Previous studies have identified eGFR slope as an independent risk factor for cardiovascular morbidity and mortality –10. Al-AThe second important finding of our study is that one calculated formula initial eGFR AUC%_12M was a better predictor of adverse renal outcomes than eGFR slope. Unlike other summary measures , both pieces of information can be captured through the use of AUC . The useAs for other approaches, such as using SE of the regression as a method of evaluating eGFR variability, our result is in agreement with Perkins RM's finding that eGFR variability by the SE did not predict ESRD outcome among CKD stage 3 patients . DespiteIn conclusion, our results demonstrate that the greater eGFR variability by AUC% is associated with the higher risk of progression to RRT. The formula of estimating eGFR variability by AUC method may provide a significant predictive value and guide the treatment strategies and choices in CKD patients.


However, the CSF biomarkers specifically mirroring Alzheimer-type brain changes, i.e., P-tau and Aβ1-42, did not contribute significantly. In healthy controls, CSF biomarkers did not explain the variance in cognitive performance. Selected CSF biomarkers of neurodegenerative processes accounted for cognitive performance in persons with bipolar disorder, but not for healthy controls. Specifically, the ratios of Aβ42/40 and Aβ42/38 were consistently associated with altered cognitive performance.The purpose of the present study was to investigate if cerebrospinal fluid (CSF) biomarkers of neurodegeneration are associated with cognition in bipolar disorder and healthy controls, respectively. CSF concentrations of total and phosphorylated tau, amyloid beta (Aβ)1-42, ratios of Aβ42/40 and Aβ42/38, soluble amyloid precursor protein α and β, and neurofilament light chain protein were analyzed in relation to neuropsychological performance in 82 euthymic bipolar disorder patients and 71 healthy controls. Linear regression models were applied to account for performance in five cognitive domains using the CSF biomarkers. In patients, the CSF biomarkers explained a significant proportion of the variance (15–36%,  The hallmark of bipolar disorder is recurrent episodes of depression and mania or hypomania . The worMeta-analyses suggest that attention/speed, memory, and executive functions are impaired in euthymic bipolar disorder , 6. An iNo biological correlates of cognitive impairment in bipolar disorder have been established.Meta-analyses of structural magnetic resonance imaging (MRI) studies report morphological differences between persons with bipolar disorder and controls , 12, butIn two recent studies, we investigated the applicability of cerebrospinal fluid (CSF) biomarkers to study neurodegenerative processes in bipolar disorder , 16. We CSF biomarkers of neurodegeneration have been linked to cognitive impairment in other disorders , 21. It The aim of this study was to evaluate potential associations between CSF biomarkers of degeneration  and cognitive function in patients with bipolar disorder. Regression models with five aggregated cognitive domains were applied using CSF biomarkers as predictors and covariates as appropriate. The models were repeated in healthy age- and sex-matched controls to determine if observed associations were disease dependent.The St. Göran Bipolar Project is a clinical longitudinal study of persons with bipolar disorder. The procedures in this project have been described in detail elsewhere . In brieThe key clinical assessment instrument was a Swedish version of the Affective Disorder Evaluation ADE), which is a standardized interview protocol developed for the Systematic treatment Enhancement Program of Bipolar Disorder (STEP-BD) . The ADEBoth the CSF sampling and the cognitive examination procedures were carried out when patients were in a euthymic mood. Euthymia was defined as MADRS  and YMRS  scores <14.Controls were included to evaluate the extent to which CSF biomarkers play a specific role in bipolar disorder. Age- and sex-matched healthy, population-based controls were randomly selected by Statistics Sweden and contacted by mail. Given an expected response rate of 1:7, seven invitations were sent out per enrolled patient. Fourteen percent of the invited controls responded to the invitation to participate, and were subjected to a preliminary telephone screening to exclude severe mental health, neurological problems, and substance abuse. Thus, 75 individuals were excluded due to drug use, no longer willing to participate, or somatic illness. Eligible persons were scheduled for a personal examination and investigated to exclude mental illness by a psychiatrist using the M.I.N.I. and selected parts of the ADE. The control subjects underwent blood-sampling, lumbar puncture, neuropsychological testing and self-rating scales. The procedures were identical in patients and controls, except that controls completed all investigations during the same day, whereas CSF sampling and the neuropsychological testing occurred at separate occasions for patients. Controls presenting potentially pathological findings were discussed between examining clinician, primary investigator, and study coordinator at case conferences. Exclusion criteria were: any current psychiatric disorder, a family history of schizophrenia or bipolar disorder in first-degree relatives, drug or alcohol abuse (based on DUDIT and AUDIT as well as serum levels of carbohydrate-deficient transferrin), neurological conditions except mild migraines, pregnancy, untreated endocrine disorders, dementia, and severe personality disorder.The study was approved by the Regional Ethics Committee in Stockholm (case no. 2005/554-31/3) and conducted in accordance with the latest Helsinki Protocol. After complete description of the study, all enrolled patients and controls consented orally and in writing to participate in the study.To reduce the risk of diurnal fluctuations, lumbar puncture was performed at 0900–1000 AM after night fasting. The spinal needle was inserted into the L3/L4 or L4/L5 interspace, and a total volume of 12 ml of CSF was drawn, gently inverted to avoid gradient effects, and divided into 1.0–1.6 ml aliquots that were stored at -80°C pending analysis. For ethical reasons, patients were not taken off their prescribed medication at the time of the sampling. All samples were thawed and refrozen once before analysis. All biochemical analyses were performed at the Clinical Neurochemistry Laboratory in Mölndal, Sweden, by board-certified laboratory technicians blinded with respect to clinical information. The CSF concentrations of sAPP α and β were determined using the MSD sAPPα /sAPPβ Multiplex Assay, while CSF Aβ38, Aβ40, and Aβ42 were analyzed using the MSD Human/Rodent (4G8) Assay , as described previously . CSF conThe administered neuropsychological test battery accords the recent recommendations from the International Society for Bipolar Disorders  coveringThe primary outcome of this study was the ability of the CSF biomarkers to explain cognitive performance in bipolar disorder. The applicability of the biomarkers to account for cognitive performance was further tested in the healthy age- and sex-matched controls.Preliminary analyses were performed to ensure no violation of the following prerequisites for regression: linearity, normality, absence of multicollinearity, and homoscedasticity. Variables that violated requirements for linearity were transformed as appropriate and variables that were highly inter-correlated were excluded for the model of interest. Linear regression was performed to assess the degree to which CSF biomarkers can explain the variance in the aggregated cognitive domain scores. CSF biomarkers were entered as independent variables in all models. The following variables were included as covariates for patients: age, sex, bipolar subtype, CGI, MADRS, YMRS, treatment with any mood stabilizer, lithium, anticonvulsants, antidepressants, antipsychotics, benzodiazepines, and anxiolytics (non-benzodiazepine anti-anxiety medication). For healthy controls, age and sex were the only applicable covariate.r2 of the model, the standardized beta values, and the p-value (two-tailed tests) of the individual variables. Analysis of variance and chi-square, where applicable, were used for group comparisons. P-values <.05 were considered significant. Alpha correction was not applied. To test if the covariates accounted for the influence of the CSF biomarkers on cognition, mediation effects were assessed using bootstrapping for continuous mediators and logistic mediation analysis for dichotomous mediators (medication). Analyses were performed using SPSS version 21  and SAS JMP version 10 .We report the adjusted The current study included 82 cases of bipolar disorder type I and II that had completed sampling of CSF and cognitive examination . The patF  = 8.7, p =. 004). Patients’ sAPPα  = 8.1, p =. 005) and sAPPβ  = 4.7, p =. 03) were lower whereas their Aβ42/38 ratio  = 4.4, p =. 03) and NFL concentrations  = 5.4, p =. 02) were higher. Patients performed worse than healthy controls in the domains of memory  = 13.6, p <.0005) and verbal functions  = 7.1, p =. 009) (Data from the St. Göran cohort on test specific cognitive performance  as well  =. 009) .2 =. 15, F = 5.0. p. 001) of the observed variance  = 4.4. p =. 006), the contribution of the CSF biomarkers NFL, Aβ42/40, and Aβ42/38 on memory remained essentially unchanged (data not shown). We also performed a separate analysis of the association between working memory and CSF biomarkers and found that the CSF biomarkers explained a significant proportion of the variance in working memory performance  = 8.0. <.0001). Low working memory performance was associated with an elevated concentration of NFL  and a higher Aβ42/38 ratio . Use of lithium was associated with increased working memory performance .As working memory can be conceived as a separate memory system , the ass2 =. 24, F = 4.01. p <.0005) of the variance in speed and attention performance. Low performance in the attention/speed domain was associated with lower concentrations of sAPPα and a lower Aβ42/40 ratio, whereas a higher Aβ42/38 ratio again was associated with higher performance. Lower age and use of anticonvulsants and antipsychotics were positively associated with speed and attention, whereas use of benzodiazepines had a negative impact. sAPPα was the only CSF biomarker with a marked impact on attention/speed . Use of benzodiazepines was negatively associated with attention (beta = -.31).In total, the model explained 24%  = 4.3. p <.0005) of the executive domain scores. Higher concentrations of T-tau explained reduced executive performance (beta = -.35). Use of anxiolytics (except benzodiazepines) was associated with increased performance, whereas benzodiazepine use was associated with reduced performance.2 =. 21, F = 3.08. p =. 002) of the variance in performance in this domain. Better verbal performance was associated with higher T-tau concentrations and a lower Aβ42/40 ratio. Higher concentrations of NFL was most strongly negatively associated with verbal performance (beta = -.39). Use of anticonvulsants was negatively associated with verbal functioning.In total, CSF biomarkers and covariates accounted for 21%  = 5.37. p = <.0005)  . Higher Mediation effects were not found for any of the covariates included in the regression models (results not displayed); CSF biomarkers and covariates contributed independently to the variance observed in the cognitive domains.CSF biomarkers did not account for any variance in cognitive performance for the controls. The only significant predictors of cognitive performance in healthy controls were sex and age for visuospatial functions  and sex This is the first study investigating the relationship between CSF biomarkers of neurodegeneration and cognition in bipolar disorder. The biomarkers independently accounted for a significant part of the individual variability of cognitive performance in the bipolar disorder group. Intriguingly, the findings could not be reproduced in the control sample. This suggests that an association with CSF neurodegeneration biomarkers and cognitive performance is not a general phenomenon. It remains to be elucidated if these associations are specific to persons with bipolar disorder, or if CSF biomarkers of neurodegeneration might also be associated with cognitive performance in other psychiatric disorders such as schizophrenia.Biomarkers and covariates best explained the variance in visuospatial functions, which is somewhat surprising as visuospatial functions are typically relatively well preserved in bipolar disorder . As the higher Aβ42/40 ratio was associated with decreased cognitive performance is therefore somewhat puzzling. However, the Aβ42/38 and Aβ42/40 ratios have also been found to increase prior to deposition of plaque in Alzheimer’s disease [In the group with bipolar disorder, higher concentration of NFL, as well as higher Aβ42/40 ratio and lower Aβ42/38 ratio, was consistently associated with decreased cognitive performance. Altered ratios of Aβ were the CSF biomarkers that accounted for most of the variance in verbal performance. Again, this finding could be related to cross-loadings on executive functions. NFL was the most important predictor of memory performance. However, the model only accounted for 15 percent of the variance in the memory domain, which dropped to 12 percent when working memory tests were removed from the memory/learning domain. Working memory alone was more strongly associated with CSF biomarkers (27%) and NFL was the CSF biomarker that contributed the most to the model. Nonetheless, this suggests that neuronal and axonal dysfunction or degeneration might contribute to reduced memory performance in bipolar disorder. Aβ40 is more adhesive than the Aβ38 isoform and thus considered more harmful . A decre disease .T-tau was associated with cognitive performance in three domains, but inconsistently so; higher T-tau concentrations was positively associated with verbal and visuospatial functions, but negatively associated with executive functions. Moreover, there was no difference in CSF T-tau between patients and controls. Hence, the value of T-tau as a predictor of cognitive performance might be limited. Also, sAPPβ was only associated with performance in one cognitive domain; lower sAPPβ concentrations correlated with decreased speed/attention performance. Furthermore, the more AD specific biomarkers P-tau and Aβ1–42 were not associated with cognitive performance in any cognitive domain. As P-tau and Aβ1–42 are established biomarkers for current and future cognitive performance in mild cognitive impairment and AD , findingA limitation of this study is that sampling of CSF was conducted on average 7 months after completion of the neuropsychological examination. Whereas the delay is not ideal, it is not likely that it has affected the results as neuropsychological performance is stable in bipolar disorder. Also, the studied CSF biomarkers are steady over a period of 2 years in patients with Alzheimer’s disease . AlthougIn conclusion, we found that CSF biomarkers of neurodegeneration were associated with cognitive performance in euthymic bipolar disorder, but not in healthy controls, in all cognitive domains independently of age, medication, disease status, and bipolar subtype. Notably, ratios of Aβ42/40 and Aβ42/38 were consistently associated with altered cognitive performance. It remains to be studied whether CSF biomarkers can also be utilized to predict changes in cognitive functioning during the course of illness.


MIF promoter variations and the risk of developing rheumatic heart disease (RHD) remain inconclusive. RHD is an important complication of streptococcal infections in the Middle East, not least in Saudi Arabia, and identifying risk markers is an important priority. Therefore, we investigated the association between two functional MIF promoter variations and RHD susceptibility and severity in Saudi patients: the MIF-173G > C substitution (rs755622) and the MIF-794 CATT5–8 tetranucleotide repeat (rs5844572). Three hundred twenty-six individuals  were genotyped using allelic discrimination and fragment analysis. Data were analyzed with respect to disease susceptibility, severity, sex, and age of onset. There was a significantly lower frequency of 173C allele carriage in RHD patients compared to controls . Interestingly, the 173C allele was associated with late disease onset (p = 0.001). The 794 5-repeat allele was associated with decreased RHD risk . In contrast, the 794 6-repeat allele was associated with increased risk of RHD . MIF promoter variations appear to have a dual role in RHD, with 173C allele non-carriers at higher risk of developing RHD at a younger age. These results require further validation in larger multi-ethnic cohorts, and functional studies are necessary to understand the underlying molecular mechanisms driving the at-risk phenotype.Although macrophage migration inhibitory factor (MIF) has consistently been shown to be an important immune modulator, data on the association between  Rheumatic heart disease (RHD) is an autoimmune disease that can develop following throat infections with one of the group A beta-hemolytic streptococci (GAS). Although standard antibiotic treatments are effective and help to prevent the disease, RHD is a common source of acquired pediatric heart disease in many developing nations , to the Of note, only some GAS strains are rheumatogenic , and onlMIF is a small gene (<0.7 kb) of three exons located on 22q11.2, and it is highly conserved across different species (Macrophage migration inhibitory factor (MIF) is a pleiotropic immune mediator important in innate and adaptive immune responses.  species . MIF exp species , resulti species , 19 and  species . Studies species . For exa species , but wheMIF promoter contains two functional variations that affect its gene expression and protein levels in vitro and in vivo: a MIF-173G > C substitution (rs755622) and a MIF-794 CATT5–8 tetranucleotide repeat (rs5844572)  , 24. Thepression . In vitrMIF promoter variations modulate the course of RHD by evaluating these sequence variants in relation to RHD susceptibility, severity, and age of onset in 326 Saudi Arabian participants.Migration inhibitory factor is pathogenic in several diseases including infectious , autoimmOne hundred and twenty-four RHD patients were enrolled in a pilot study at the Pediatric Cardiology Clinic at the Maternity and Children Hospital, Al-Madinah region, Saudi Arabia between March 2013 and June 2014. The Maternity and Children Hospital ethics committee approved the study protocol, and the authors followed the norms of the World Medical Association Declaration of Helsinki. All adult patients and donors or the parents or guardians of child participants (<18 years old) signed a fully informed and written consent form approved by the ethics committee.via the National Blood Service in the Al-Madinah region. Controls were age-, gender-, and ethnically matched unrelated volunteers with no history of cardiac or autoimmune diseases. All participants were of Saudi Arabian ethnicity.The patient and control cohorts are described in previous studies , 16. BriMIF-173 polymorphism was genotyped using the duplex quantitative TaqMan 5′ Allelic Discrimination Assay  as directed by the manufacturer. Briefly, assays were performed in a final volume of 10 μl  in 96-well plates using the following amplification protocol: 95°C for 10 min followed by 50 cycles at 95°C for 15 s and 60°C for 1 min . Fluorescence detection took place at 60°C. Non-template controls were included in each run. The genotype call rate was over 99%. Duplicate genotyping of 10% of samples selected at random was performed for quality control. Assays were performed using the StepOnePlus system, and the automated Sequence Detection Software (SDS) v2.3 was used for auto-calling (Applied Biosystems).DNA was purified from 2-ml whole blood using the QIAamp DNA Mini Kit  according to the manufacturer’s protocol. Extracted DNA was quantified by spectrophotometry  and stored at −20°C until use. The MIF-794 repeat was genotyped by conventional PCR, and the product was separated by capillary electrophoresis . Reactions were performed with 10 pmol of each primer: forward primer (FAM-5′-AAATCTCTGAGGACCTGGCC-3′) and reverse primer (5′-CACCGTGTATGGCCTCTCAT-3′) designed using Primer3.2, 10× PCR buffer, dNTPs, and 10 units of Taq DNA polymerase . Samples were amplified using the Veriti thermal cycler . The cycling and amplification conditions were initial denaturation at 95°C for 2 min followed by 35 cycles with denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s. Automated capillary electrophoresis on the 3500 Genetic Analyzer was performed for each sample following the manufacturer’s protocol, and the CATT alleles were identified using GeneMapper v4.1 software (Life Technologies).The MIF promoter polymorphisms between cases and controls were tested using chi-squared contingency tables or Fisher’s exact test as appropriate. Linear regression was used to test for any association between the genetic variations and age of onset . Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. A p-value <0.05 was considered statistically significant.Statistical analyses were conducted using SPSS version 17 . In addition, results were confirmed using a freely available online statistical tool, VassarStats.MIF-173 (rs755622) and MIF-794 (rs5844572) polymorphisms. RHD patients were 56% males and 44% females, and 67 patients (54%) had MVL and 57 patients (46%) had CVL. Carditis (64%) and arthritis (57%) were present in the majority of patients. Acute phase reactants were elevated in 79% of patients at presentation . Likewise, 173C allele carriage (CC + GC vs. GG) was associated with a decreased risk of developing RHD .The MIF-794 CATT5–8 repeat variation in RHD and healthy subjects are shown in Table 5-allelic frequencies were significantly higher in the control group than RHD patients . On the other hand, 794 CATT6-allelic frequencies were associated with an increased risk of developing RHD . Most evidence indicates that 794 CATT6–8 repeats have higher transcriptional activity than 794 CATT5 repeats; therefore, we grouped 794 CATT6–8 repeats and termed them the X allele. X-allele frequency was more common in RHD subjects than in healthy controls .The genotypic and allelic frequencies for the MIF-173C allele carriers than non-MIF-173C allele carriers in RHD patients . We report that the pleiotropic inflammatory cytokine MIF exerts dual effects in RHD patients. The 173C allele was associated with reduced RHD risk and, interestingly, this genotype was associated with later disease onset. In contrast, the 794 CATT6 allele was associated with increased RHD risk, but the 794 CATT5 allele was associated with reduced RHD risk. Genetic variability in MIF may have a dual impact on the development of RHD in the Saudi Arabian population.In this pilot case–control study, the impact of MIF alleles on the immunopathogenesis of infectious and autoimmune diseases have been documented. For example, in SLE, MIF alleles associated with higher gene expression  are associated with a lower risk of SLE  development in SLE, supporting a protective role for MIF in SLE , thereby providing metabolic protection (To our knowledge, this is the first case–control study of the impact of sed risk . However disease . In myoc disease . AMPK photection . Howeverotection . In RHD,otection . In turnMIF-173C allele was associated with later disease onset as well as decreased risk of RHD. To our knowledge, this is the first study to examine the effect of polymorphisms on the age of onset of RHD. These data suggest that MIF action may be age dependent; however, the results must be considered with caution since there are different definitions of “age of onset,” and advanced diagnostic echocardiography has influenced RHD burden data (Of particular note, the den data , 48. Sevden data  and highden data . In animden data . Interesden data . ClearlyMIF promoter variations and RHD. These findings require replication in larger, multi-ethnic studies to overcome some of the ethnic bias in our study, the retrospective analysis, and to provide sufficient power for robust subgroup analysis. Furthermore, we only analyzed two polymorphisms and not the entire MIF gene, and it would be useful to analyze other MIF polymorphisms and functional genetic variations in downstream signaling pathways to fully elucidate the function of this pathway in RHD. Finally, studies from Arab countries are scarce with respect to MIF variations in any infectious or autoimmune diseases, making ethnic comparisons difficult and only permitting comparisons with results from non-Arab populations.Here, we report an association between MIF promoter variants may have an impact on RHD susceptibility, severity, and age of onset. Further prospective, multi-ethnic studies are required.In conclusion, AA designed the study, planned and performed experiments, analyzed data, and wrote the manuscript. NA-H and AA-M collected patient samples and clinical data and contributed to research discussion. AE and WA-H analyzed data and edited the manuscript. YA and KA-H designed the study, contributed to research discussion, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SD (n = 150) rats were randomly divided into three groups of normal, CCl4 model and rhCygb groups. After model establishment, rats in rhCygb groups were administered daily with rhCygb . Histological lesions were staged according to metavir. Serum parameters including ALT, AST, HA, LN, Col III and Col IV were determined. The liver proteins were separated by 2-DE and identified. As a result, the stage of hepatic damage and liver fibrosis in rhCygb groups were significantly milder than that in CCl4 model groups. Meanwhile, rhCygb dramatically reversed serum levels of ALT and AST, and also markedly decreased the liver fibrosis markers levels of LN, HA, Col III and Col IV. In 2-DE, 33 proteins among three groups with the same changing tendency in normal and rhCygb treated groups compared with CCl4 model group were identified. GO analysis showed that several identified proteins involved in oxidative stress pathway. The study provides new insights and data for administration of rhCygb reversing CCl4-induced liver fibrosis suggesting that rhCygb might be used in the treatment of liver fibrosis.This study aims to investigate whether the use of recombinant human cytoglobin (rhCygb) impact on hepatic fibrogenesis caused by CCl Liver fibrosis, a pathological process common to chronic liver diseases, is caused by a range of actors, and has a highly deleterious impact on human health. It is well-established that liver fibrosis formation results from an excessive accumulation of extracellular matrix proteins including collagen, a process that occurs in most types of chronic liver disease2Because of the urgent clinical need, the development of an anti-fibrotic drug would be an auspicious moment in medical research. Up to now, several candidate treatments with anti-fibrotic properties have been developed, which variously act by blocking inflammatory pathways467911in vitro and in vivo, to protect hepatic stellate cells (HSC) from oxidative stress and suppress their differentiation to a myofibroblast-like phenotype161719204-induced liver fibrosis in rats at various stages of the disease’s progression, from mild to severe fibrosis.The Cygb protein was first discovered in 2001 by proteomic analysis in rat stellate cells, and was initially named stellate cell-activated protein (STAP)E. coli BL21 (DE3). For expression, the bacteria cultures were induced for 4 h using 1 mM isopropyl β-d-thiogalactoside (IPTG) after the culture OD 600 has reached 0.6. The total bacteria cells were collected by centrifugation at 5,000 rpm for 10 min, resuspended in PBS, and broken down by sonication. The rhCygb was purified by sephadex G-25 and affinity chromatography which the monoclonal antibody was prepared by our laboratory. The purified protein was analyzed with SDS–PAGE and identified by mass spectrometry with ABI 4700 Proteomics Analyzer . The yields of rhCygb measured by the software BandScan 5.0 based on SDS-PAGE results. The total antioxidation capacity (T-AOC) of rhCygb was measured spectrophotometrically as described by Tang et al.Human cytoglobin cDNA was obtained from HepG2 cells by RT-PCR. A recombinant construct of pET28a-hCyt were transformed into All experimental procedures were conducted in conformity with institutional guidelines approved by the Chinese Association of Laboratory Animal Care. All experimental protocols were approved by the Institutional Animal Care and Use Committee in Southern Medical University, Guangzhou, China.Sexually mature sprague–dawley (SD) rats (n = 150) weighing 180–220 g were obtained from the experimental animal center of Southern Medical University. Care of the animals used in this investigation was conducted according to the guidelines approved by the Chinese Association of Laboratory Animal Care. Rats were housed, five per cage, under constant temperature (20 ± 2 °C) and humidity (70%) with a 12 h light/dark cycle and with free access to chow and water.in vivo using the procedure approved by Chen et al.BALB/c mice of both sexes, weighing 18–24 g, were obtained from the experimental animal center of Southern Medical University. The acute toxicity of rhCygb was determined 4) administration at three different dosage levels:4 in paraffin oil  was administered by subcutaneous injection twice a week (Tuesday and Friday) for 10 weeks;Mild model group: 2 ml/kg of 25% CCl4 treatment for 3 more weeks;Medium model group: treatment in group (a) plus 50% CCl4 for 2 more weeks.Severe model group: treatment in group (b) plus 62.5% CClThree degrees of liver fibrosis were induced by chronic carbon tetrachloride , CCl4 (model group) or rhCygb  in combination with CCl4 for 12 weeks. At the end of the experiments, all rats were sacrificed within 24 h after the last injection.To evaluate the effect of rhCygb on liver fibrosis, 30 rats were used in each of the groups . A schedule of the treatment is shown in Venous blood samples were collected for serum separation. Samples were stored at −20 °C until further analysis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured spectrophotometrically by Olympus/AU 5200 .Serum liver fibrosis markers hyaluronic acid (HA), laminin (LN), collagen III (Col III) and collagen IV (Col IV) were determined using enzyme-linked immunosorbent assay (ELISA) kits .et al.In the end of the experiments, rats liver were removed and rinsed with cold saline water. Then liver lesions were fixed, embedded, and stained with Sirius red and hematoxylin-eosin (HE)4 model group and rhCygb treated group (medium). The samples were then frozen in liquid nitrogen and homogenized with a mortar and pestle. Proteins were prepared in lysis buffer. A total of 120 μg of protein was separated by two-dimensional electrophoresis (2-DE) according to the product manual ‘2-DE for Proteomics’ . After silver staining, each spots were cut out from the gel and digested using trypsin according to the method described by Qualtieri et al.Five liver samples (approximately 30–40 mg wet) were removed from vehicle group, CClhttp://www.matrixscience.com/)p < 0.05).Peptide mixtures of each gel spot were dissolved in 0.1% TFA, desalted, and concentrated. The samples were then mixed with the same volume of matrix (CHCA in 30% ACN/0.1% TFA), spotted on a target disk, and allowed to air-dry. Samples were analyzed using a Bruker ultrafleXtreme MALDI-TOF mass spectrometer . The protein database search was performed using the MASCOT search engine (https://david.ncifcrf.gov/)http://genecodis.cnb.csic.es/)To identify significantly represented biological themes and functional groups in the protein list , the genet al.Modified gene set enrichment analysis (GSEA) was used to assess functional significance at the level of sets of genes as previous described by Li All data were expressed as the mean ± standard deviation from at least 5 independent experiments. Figures were obtained from at least 5 independent experiments with similar patterns. The biochemical experimental data were analyzed by two-way ANOVA. The Kruskal-Wallis was used for comparisons. Statistical analyses were performed using SPSS software version 19.0 .p < 0.01) (The purity of rhCygb was 95% by BandScan 5.0 software. The antioxidant activity of rhCygb is 95.58 ± 2.67 U/mg ( < 0.01) .When given as a single dose of 100 and 500 mg/kg, rhCygb produced no signs of acute toxicity in the 14-day period of observation. No death was recorded in the 100- and 500-mg/kg-treated group. All groups of mice exhibited stable temperature values throughout the period of observation.4 were sacrificed at 3 different times, 10th week, 13th week and 15th week. At sacrifice, all rats presented marked fibrosis with significant differences in the amount of liver fibrotic tissue among groups. Hepatolobular injury was observed with centrilobular necrosis, balloon cells, and lipids accumulation in 15-weeks-model rats (4) for 10 weeks in SD rats, administration of CCl4 significantly (p < 0.01) increased the concentration of AST, ALT, LN, HA, Col III and Col IV in rat serum compared with the levels in the vehicle group (4 (50% CCl4) for a further 3 weeks in group b (medium) resulted in a marked prolongation of toxicity, increasing (p < 0.01) serum enzyme values about two-fold except for Col IV. However, there was no significant (p > 0.05) difference between the group b (medium) and group c (severe).All experiments successfully induced fibrosis, as determined by the examination of liver histology and serum biochemistry . The mordel rats . The samdel rats . At the le group . Moreove4 administration. Rats were treated with rhCygb  in combination with CCl4  for 12 weeks. All of the rats survived during the experimental period. The influence of CCl4 and rhCygb on liver was depicted in 4 reversed dramatically by rhCygb. As shown in 4-administered rats significantly changed, especially in the medium and severe groups. These groups, after treatment with rhCygb, exhibited a clear and marked improvement in their liver, when compared with those of the CCl4-administered groups. Photomicrographs of hepatic specimens stained with Sirius red were depicted in 4 model groups. As expected, the high CCl4 treatment caused fibrous connective tissue proliferation as well as a good deal of collagen fiber formation. Moreover, increased dosages caused even more frequent pseudo lobules to appear in model control groups. The hepatic fibrosis score in the CCl4 groups increased to 3.40 ± 0.33, 3.80 ± 0.13 and 3.90 ± 0.1, respectively. In the rhCygb treated groups, liver had little collagen fiber formation in the peripheral area, and no pseudo lobules. The positive area of Sirius red staining was significantly lower than that of the CCl4 groups. The scores were 0.27 ± 0.58 (mild group), 0.8 ± 0.19 (medium group) and 0.57 ± 0.16 (severe group), respectively. Similar observations were made in rhCygb groups which evinced statistically significant lower fibrosis scores compared to CCl4 groups.We next evaluated the effect of rhCygb on liver fibrosis following chronic CCl4 model groups compared with normal control groups, reflecting hepatocelluar damage in CCl4-induced liver fibrosis. Then rhCygb treatment significantly (p < 0.01) reversed serum ALT and AST levels in 10 + 12 weeks, 13 + 12 weeks and 15 + 12 weeks groups, to the levels almost equal to vehicle controls.The levels of serum ALT and AST are markers of liver damage. As shown in 4 groups were higher (p < 0.01) than controls  liver fibrosis marker levels of LN, HA, Col III and Col IV in 10 + 12 weeks, 13 + 12 weeks and 15 + 12 weeks groups.In this study, serum LN, HA, Col III and Col IV levels in CClcontrols . Compare4-induced fibrotic liver, liver tissues were harvested from the (13 + 12)-weeks-old control group, the CCl4 model group, and rhCygb treatment group of rats. When examined, the 2-DE gel showed an obvious separation of liver tissue proteins, with 1312 proteins in the vehicle group, 1325 proteins in the CCl4 model group and 1259 proteins in rhCygb group. Comparing the spots of vehicle to model group or model to rhCygb group by PDquest software for image analysis, a total of 75 protein spots exhibited significant (p < 0.05) differences in protein expression between vehicle and model or between model and rhCygb group . A total of 33 peptide mass fingerprints (PMFs) were matched and identified by MALDI-TOF-MS and protein database searching, as displayed in To study the influence of rhCygb on the proteome of CClgb group . These 7gb group : patternWe analyzed the GO enrichment of these upregulated and downregulated proteins . Relevanst, p = 3.33 e−8, FDR = 2.27 e−4), or in the genes highly expressed in hepatocellular carcinoma with good survival . FDR q-value was considered significant at < 0.05.GSEA  revealedMuch experimental evidence and clinical studies has shown that the physiological functions of Cygb are associated with various diseases, such as cancer4 proved to be highly useful as an experimental model for the study of liver damage in humans3334364. The results of the present study revealed that continuous and increasing doses of CCl4 resulted in advanced fibrosis which caused an elevation of levels of serum marker enzymes such as ALT, AST, HA, LN, Col III and Col IV from the mild to the severe model. The serum marker enzymes ALT and AST have previously been reported as important diagnostic factors for hepatic diseases, as they are cytosolic enzymes of the hepatocyte, and increased activity in their circulation reflects cell damage and leakageCCl4 model groups increased markedly compared with normal groups. However, after the treatment of rhCygb (therapy but not removing CCl4), we detected that the serum contents of HA, LN, Col III and Col IVdecreased to the level of normal groups. This may be the basic mechanism of rhCygb reversing rat hepatic fibrosis.In addition, HA, LN, Col III and Col IV have been found to be ideal serum markers of hepatic fibrosis, which play an important role in detecting the degree of hepatic fibrosis4 model groups: 70% in the mild group, 80% in the medium group and 90% in the severe group. However, score 4 didn’t show up in any of the rhCygb treated groups. In time, score 0 was present in 24 rats in the mild group, 18 rats in the medium group, and 19 rats in the severe group. These data are in solid agreement with the activities of serum biomarkers. Interestingly, the stages of fibrosis were previously believed to be progressive and largely irreversible. There is now mounting experimental and clinical evidence suggesting that liver fibrosis can regress in all chronic liver diseases by withdrawing the cause of liver injury or by treatment of the disease414-induced rat liver fibrosis was markedly reversed by administration of rhCygb. He et al. reported the similar findings in their use of rhCybg in the thioacetamide (TAA)-induced rat liver fibrosis modelIn response to liver injury induced by whatever cause, collagen and matrix proteins are produced in much higher quantities by activated hepatic stellate cells, which ultimately leads to liver fibrosis4-induced rat model. Thirty-three differentially expressed proteins appeared among the three groups, with the same alteration profile in the vehicle and rhCygb treated groups compared with the CCl4 model group, identified through comparative proteomics. Functional annotation analysis showed several identified proteins involved in oxidative stress pathways, such as the response to chemical stimuli, oxidative-reduction, and xenobiotic metabolic processes. Oxidative stress is thought to be a critical factor in the development of CCl4-induced liver fibrosis44 model group, whereas in the rhCygb-treated group they were up-regulated (as they were in the vehicle group). These findings show that impairment of fatty acid oxidation may contribute to hepatic fibrosis.We systematically analyzed the effect of rhCygb on liver proteome in the CClIn summary, the results of the present study demonstrate that in addition to the effect of reversing liver fibrosis, rhCygb could reduce the serum levels of ALT, AST, HA, LN, Col III and Col IV, indicating that it has definite effects in reversing the formation of liver fibrosis in the course of chronic hepatitis. The differentially expressed proteins have several groups of biological functions, especially relating to oxidative stress, which are helpful in revealing the underlying mechanisms of rhCygb action against liver fibrosis. rhCygb could be a novel candidate drug in combating liver fibrosis.How to cite this article: Li, Z. et al. The Effect of rhCygb on CCl4-Induced Hepatic Fibrogenesis in Rat. Sci. Rep.6, 23508; doi: 10.1038/srep23508 (2016).