Based on the principles of the in vitro staining technique, hypotonic swelling test, and water test, the Eosin Y-water test method was developed to simultaneously detect the integrity of the sperm head and tail and sperm membrane structure and function. As a widely used method in clinical laboratories in China, the Eosin Y-water test is methodologically characterized by three advantages. Firstly, both the sperm head and tail can be detected at the same time, which allows easy and comprehensive assessment of membrane damage in different parts of sperm. Secondly, distilled water is used instead of the usual formula solution to simplify and standardize the test by eliminating any potential effects on the water molecules through the sperm membrane due to different osmotic pressure or different sugar proportions and electrolyte solutions. Thirdly, the test takes less time and thus can be repeated before and after treatment. This article focuses on the fundamental principles and modification of the Eosin Y-water test and its application in sperm function examination and routine semen analysis for male infertility, assessment of the quality of sperm retrieved by testicular fine needle aspiration, semen cryopreservation program development, and evaluation of sperm membrane integrity after microwave radiation.
Cell Membrane ; China ; Cryopreservation ; Eosine Yellowish-(YS) ; Fluorescent Dyes ; Humans ; Infertility, Male ; diagnosis ; Male ; Osmotic Pressure ; Semen Analysis ; methods ; Sperm Head ; Sperm Motility ; Sperm Tail ; Spermatozoa ; Staining and Labeling ; Water

Objective: In order to evaluate the reliability and feasibility of pueraria reference extractive substance (RES) used in biological sample, the pharmacokinetics of 3′‑hydroxy puerarin (3′-HP), puerarin, 3′‑methoxy puerarin (3′-MP), and daidzein-8-C-apiosyl-(1-6)-glucoside (DAG) in beagle plasma following oral administration of Yufeng Ningxin Tablet were quantitated. Methods: A reliable and sensitive high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) method developed with chromatographic separation was operated on a Merck C18 column, and acetonitrile-5 mmol/L ammonium was used as mobile phase in gradient elution. The plasma samples were deproteinized by acetone, detected by triple quadrupole mass spectrometry with an electrospray ionization interface, and quantified using selected ion monitoring mode. The pharmacokinetic parameters were calculated by Winnonlin 4.1. Results: The calibration curves of the reference extractive substance and standard substance methods were linear over the ranges 0.0417–11.3309 µg/mL and 0.0394–10.0000 µg/mL. The intra-day and inter-day precision of the two methods at three concentrations were less than 13.63%, and the average recoveries of 3′-HP, puerarin, 3′-MP, and DAG were more than 70.67%. The RSD of the mean plasma concentrations of the analytes calculated by the two methods was less than 5%, and cos (ϑ) = =1.000. Among the analytes, puerarin showed the highest blood concentration [(940 ± 185) ng/mL] and the longest retention time [(5 ± 1) h] in the dog's bodies. Conclusion: Pueraria reference extractive substance can be seen as an alternative to the standard substance to overcome the scarcity of standard substance for the analysis of biological samples.

To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro. The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1. Expression of fusion protein was directed by the tac promoter. The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction. The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography. GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained. The GST-KGDX was expressed in E. coli to a level of 48.02% of total cellular protein. GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01). In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa. Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST. It is concluded that the GST-KGDX fusion protein can be produced by E. coli and used as an antiplatelet agent.
Adult ; Escherichia coli ; genetics ; Female ; Fibrinogen ; metabolism ; Flow Cytometry ; Glutathione Transferase ; pharmacology ; Humans ; Male ; Oligopeptides ; pharmacology ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; isolation & purification ; pharmacology ; Platelet Glycoprotein GPIIb-IIIa Complex ; antagonists & inhibitors ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification ; pharmacology

BACKGROUNDMolecular hydrogen, as a novel antioxidant, has been proven effective in treating many diseases. This study aimed to evaluate the therapeutic effects of hydrogen saturated saline in treatment of a rat model of diabetes mellitus and a rat model of insulin resistant.

METHODSA rat diabetes mellitus model was established by feeding a high fat/high carbohydrate diet followed by injection of a small dose of streptozotocin, and an insulin resistant model was induced with a high glucose and high fat diet. Hydrogen saturated saline was administered to rats with both models conditions on a daily basis for eight weeks. A pioglitazone-treated group and normal saline-treated group served as positive and negative controls. The general condition, body weight, blood glucose, blood lipids, and serum insulin levels of rats were examined at the 8th week after treatment. The oxidative stress indices, including serum superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) were also evaluated after eight weeks of treatment using the commercial kits.

RESULTSHydrogen saturated saline showed great efficiency in improving the insulin sensitivity and lowering blood glucose and lipids. Meanwhile, the therapeutic effects of hydrogen saturated saline were superior to those of pioglitazone. Hydrogen saturated saline markedly attenuated the MDA level and elevated the levels of antioxidants SOD and GSH.

CONCLUSIONHydrogen saturated saline may improve the insulin resistance and alleviate the symptoms of diabetes mellitus by reducing the oxidative stress and enhancing the anti-oxidant system.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; Hydrogen ; therapeutic use ; Hypoglycemic Agents ; therapeutic use ; Insulin Resistance ; Oxidative Stress ; drug effects ; Rats ; Sodium Chloride ; chemistry ; Thiazolidinediones ; therapeutic use

OBJECTIVETo investigate the anti-hypoxia and anti-oxidation effects of aminophylline on human with acute high-altitude exposure.

METHODSTotally 100 young male army members newly recruited from Sichuan province (400 meters above sea level) were enrolled. They were randomly divided into two groups: 50 in aminophylline group (A group) and 50 in control group (C group). A group and C group orally took aminophylline and placebo respectively for 10 days, 7 days before entering Lhasa (3 658 meters above sea level) by air and 3 days after it. Several parameters were measured at three time points: before drug taken, 7 days after drug taken, and 3 days after ascending high altitude. These parameters included serum levels of nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), hydrogen dioxide (H2O2), lactic acid (LA), as well as arterial oxygen saturation (SO2), arterial oxygen partial pressure (PaO2), and arterial carbon dioxide partial pressure (PaCO2). Statistical analysis was conducted to compare the difference between two groups with Stata 7.0 software system.

RESULTSThere were no statistical differences between groups in hypoxia and oxidation indicators before and after drug taken in plain area. Three days after ascending high altitude, the serum levels of SOD, CAT, H2O2, LA, PaCO2 increased in both groups, yet to a much larger degree in C group than A group (P < 0.01); and NO, SO2, PaO2 decreased more markedly in C group (P < 0.05 for NO, P < 0.0001 for SO2 and PaO2).

CONCLUSIONAminophylline has significant anti-hypoxia and anti-oxidation effects at high altitude.

Acute Disease ; Adolescent ; Altitude Sickness ; drug therapy ; prevention & control ; Aminophylline ; therapeutic use ; Carbon Dioxide ; blood ; Catalase ; blood ; Humans ; Nitric Oxide ; blood ; Oxygen ; blood ; Partial Pressure ; Superoxide Dismutase ; blood ; Young Adult

Objective:To evaluate the relationship between preoperative plasma trimethylamine oxide (TMAO) concentrations and postoperative delirium (POD) in elderly patients.Methods:One hundred and eighty patients, aged ≥65 yr, of American Society of Anesthesiologists Physical Status classification Ⅱ or Ⅲ, with body mass index of 17-30 kg/m 2, scheduled for elective hip replacement, were enrolled in this study. Venous blood samples were collected at 1 day before surgery, and the plasma TMAO concentrations were measured by high throughput liquid chromatography-tandem mass spectrometry. The patients were divided into 3 groups according to the concentrations of TMAO: low-concentration TMAO group (group L, ≤1.2 μmol/L), moderate-concentration TMAO group (group M, 1.3-1.9 μmol/L), and high-concentration TMAO group (group H, ≥2.0 μmol/L). All patients received superior inguinal fascia iliaca compartment block combined with intravenous general anesthesia. POD was identified by the Confusion Assessment Method-Chinese Reversion scale at 1, 2, 3 and 7 days after operation. The patients were divided into POD group and non-POD group according to whether POD occurred. Binary logistic regression analysis was used to evaluate the relationship between different plasma TMAO concentrations and POD. The receiver operating characteristic curve was used to evaluate the efficacy of plasma TMAO concentrations in predicting the occurrence of POD. Results:There were 60 cases in L group, 67 cases in M group and 53 cases in H group, and the incidence of POD was 12%, 22% and 32% in L, M and H groups, respectively. The incidence of POD was significantly higher in group H than in group L ( P<0.05). The plasma TMAO concentration was significantly higher in POD group than in non-POD group ( P<0.05). Logistic regression analysis showed that the risk of POD was 3.91 times higher in group H than in group L ( P<0.05). The area under the receiver operating characteristic curve of preoperative plasma TMAO concentrations in predicting POD was 0.754 (95% confidence interval 0.671-0.838, P<0.05). When the Youden index was 0.426, the optimal cut-off value of plasma TMAO concentrations was 1.625 μmol/L, and the specificity and sensitivity were 0.795 and 0.631, respectively. Conclusions:Elevated preoperative plasma TMAO concentration is associated with an increased risk of POD in elderly patients, and it has a good predictive value for POD.

Objective To explore the real life experience of family caregivers among mild cognitive impairment (MCI) patients during health care so as to provide a reference for reducing family caregivers burden.Methods From March 2017 to March 2018,we selected 18 family caregivers of community MCI patients in Department of Neurology,rehabilitation and memory outpatient at the Affiliated Hospital of Medical School of Ningbo University as subjects by purposive sampling.Semi-structured interview,a phenomenological approach of qualitative research,was used to collect data,and Colazzi analysis was used to sort out,analyze data and refine theme.Results A total of four themes in real life experience of family caregivers of community MCI patients were extracted including family caregivers lacking knowledge about disease (fear,worry and stigma),family caregivers being with high pressure during health care,compromising and confronting actively,expecting to seek more social supports (eager to learn disease-related knowledge,hope to get relatives' understanding and help,gain community supports and environmental supports).Conclusions Community MCI health care brings negative influences on life of family caregivers.Medical staff,family members and community organization need to provide multi-path support to reduce mental pressure and social economic burden of family caregivers of community MCI patients so as to improve their quality of life.

Objective To evaluate the feasibility and safety of stent implantation in treating patients with symptomatic vertebral artery kinking.Methods Thirty-six patients with vertebral artery kinking confirmed by DSA and accorded with the stent implantation indications were chosen in our study;these patients were performed stent implantation.The stenosis and haemodynamics changes,and recent and postoperative complications were observed; Malek scale was adopted to evaluate the clinical efficacy during the 1-year follow-up.Results The success rate of stent implantation for patients with symptomatic vertebral artery kinking was 100％.The percentage of stenosis was significantly reduced from(76％±15％)to(8％±3％)after stenting(P＜0.05); the mean flow velocity of kinking lesion was obviously decreased from([45.4±22.3]cr/s)to([31.8±15.1]em/s,P＜0.05); cerebellum hematoma was noted in 1 patient subcutaneous hematoma of puncture point in 2 and femoral artery pseudoaneurysms after the stenting in 1; all these complications did not cause serious consequences.Malek scale indicated that 31 patients got 1 point,3 got 2 points,1 got 3 points,1 got 4 points and no one got 5 points; 31patients were asymptomatic during the 1-year follow-up; no significant in-stent restenosis was found.Conclusion Symptomatic vertebral artery kinking can be treated safely and effectively with stent implantation.

ObjectiveTo observe the morphological changes of bone in the progress of chronic fluorosis.MethodsWistar rats were randomly divided into three groups,30 rats in each group:normal control group,experimental group Ⅰ and experimental group Ⅱ according to body weight.Rats in normal control group drank distilled water freely.Experimental group Ⅰ and group Ⅱ drunk distilled water with sodium fluoride preparation of fluorine containing ion 100,150 mg/L solution for six months,respectively.Bone mineral density was detected by X-ray,bone morphological changes were observed under light microscope and bone histomorphometric parameters were calculated using image analysis software.ResultsThe bone mineral density values were different statistically between the three groups after feeding for 2 and 4 months(F =19.79,3.28,all P ＜ 0.05).However no significant difference was found after feeding for 6 months(F =1.80,P ＞ 0.05).The bone mineral density of experimental group Ⅰ (0.20 ± 0.03,0.21 ± 0.03) was significantly higher than that of the normal control group(0.17 ± 0.03,0.20 ± 0.04) after feeding for 2 and 4 months.The bone mineral density of experimental group Ⅱ (0.21 ± 0.02) was lower than that of normal control group(0.22 ± 0.03) after feeding for 6 months.The bone lamella in experimental group Ⅰ was arranged disorderly,the number of osteocytes increased with their nucleus atrophy and the osteoblasts were more than that of control grouo which arranged in layers observed under light microscooy.In exoerimental group Ⅱ,the bone lamella was bent deformation,the number of osteocytes had decreased with their nucleus shrinking or even disappeared and the number of osteoclasts had increased significantly observed under light microscopy.In experimental group Ⅰ,the mean trabecular density [(0.33 ± 0.03)％] increased and the mean trabecular separation,thickness [( 163.57 ± 1.99),(59.26 ± 7.18 ) μm] decreased compared with that of normal control group [(0.31 ± 0.02)％,(186.60 ± 2.90)μm,(86.42 ± 1.48)μm,all P ＜ 0.05].In experimental group Ⅱ,the mean trabecular density[(0.26 ± 0.02)％] decreased,the mean trabecular thickness[(71.42 ± 10.77)μm] reduced compared with that of normal control group[(0.31 ± 0.02)％,(86.42 ± 1.48)μm].ConclusionsExcess fluoride can damage bone tissue.Low doses of fluoride can stimulate osteoblast activity and enhance osteogenesis.The activity of osteoblasts is great than that of osteoclasts.High doses of fluoride can stimulate both osteoblasts and osteoclasts activity,but mainly the activity of osteoclasts,and bone resorption increases.

ObjectiveTo detect the concentration and distribution of fluoride ions in osteoblasts exposed to fluoride in vitro culture,and to provide basic information for studying the effect of fluoride on osteoblast injury.MethodsIn vitro cultured osteoblasts were exposed to 0,5,10,20,40 mg/L fluoride for 3,10,30 d (n =6),respectively.Concentration and distribution of fluoride ions in the cytoplasm and the nucleus of these osteoblasts were determined by nuclear magnetic resonance spectroscopy.Results(①) After cultured for 3 d,fluoride ion content of the bone cytoplasm exposed to different concentrations of fluoride 0,5,10,20,40 mg/L were (0.83 ±0.65),(0.54 ± 0.23),(0.65 ± 0.77),(0.59 ± 0.87),(3.64 ± 1.21 )mg/L,respectively,and the values of exposed to 40 mg/L fluoride group was significantly higher than that of exposed to 0,5 mg/L groups (all P ＜ 0.05).(②)after cultured for 10 d,the composition of the fluoride ion in cytoplasm of exposed to fluoride 10,20,40 mg/L groups were (4.03 ± 1.23),(3.66 ± 0.98),(6.26 ± 2.10)mg/L,respectively,which were higher than that of exposed to 0,5 mg/L groups [(0.78 ± 0.75),(2.69 ± 0.89)mg/L,respectively,all P ＜ 0.05].Of fluoride 20,40 mg/L groups,the composition of the fluoride ion in nucleus were (1.63 ± 1.19),(2.17 ± 1.21 )mg/L,respectively,which were higher than that of 0,5 mg/L groups[(0.65 ± 0.46),(1.57 ± 0.33) mg/L,all P ＜ 0.05].(③)After cultured for 30 d,of the exposed to fluoride 10,20,40 mg/L groups,the composition of the fluoride ion in cytoplasm were (3.99 ± 0.84),(4.33 ± 1.67),(5.80 ± 1.38)mg/L,respectively,which were higher than that of 0,5 mg/L groups[(0.88 ± 0.44),(2.84 ± 0.43)mg/L,all P ＜ 0.05].The composition of the fluoride ion in nucleus of the fluoride 20,40 mg/Lgroups were (3.33 ± 1.46),(3.53 ± 1.22)mg/L,respectively,which were significantly higher than that of 0,5mg/L groups [(0.70 ± 0.66),(1.99 ± 0.76)mg/L,all P ＜ 0.05].ConclusionsWhen osteoblasts are exposed to fluoride environment,fluoride ions enter into the osteoblasts quickly,and quickly accumulate in the nucleus,showing a special affinity between fluoride and bone tissue.Intracellular fluoride ions increase with the increase of contact time and exposure dose.