Simultaneous quantitative analysis of different ceramide species in cells by high-performance liquid chromatography-tandem mass spectrometry / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a high-performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS) method for simultaneous quantitative analysis of different ceramide species in cells.</p><p><b>METHODS</b>The analysis was performed on an Agilent 1290 HPLC system with a ZORBAX Eclipse XDB-C8 (150.0 mm × 2.1 mm, 3.5 μL) column and a temperature of 35 ℃. Methanol with 1 mmol/L ammonium formate and 0.2% formic acid was used as mobile phase A and 100% methanol was used as mobile phase B. And the ceramides were separated by gradient elution at a flow rate of 0.3 mL/min. Electrospray ionization (ESI) with multiple reaction monitoring (MRM) was used in the analysis.</p><p><b>RESULTS</b>Four ceramide species all had a good linear response in the determination ranges (R² ≥ 0.9987). The average recoveries (n = 9) were 99.1%,99.9%,100.5% and 98.2% with RSDs of 5.6%, 5.1%, 4.7% and 5.5%, respectively. In addition, the levels of ceramides in FL cells were relatively stable, while the C24-ceramide had the highest level.</p><p><b>CONCLUSION</b>The HPLC-ESI-MS method for simultaneous analysis of ceramides has high accuracy, reproducibility and linearity, which may be used for quantification of ceramide species in cells.</p>

The effects of extremely low frequency electromagnetic field exposure on the pH of the adult male semen and the motoricity parameters of spermatozoa in vitro / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the effects of 50-Hz extremely low frequency electromagnetic field (ELF-EMF) exposure on the pH of the adult male semen and the motoricity and motoricity parameters of spermatozoa.</p><p><b>METHODS</b>Healthy adult male fresh semen was exposed to a 50-Hz EMF at 0.4 mT for 15, 30 and 60 min, respectively. The pH value of the semen, the motoricity and motoricity parameter of spermatozoa were detected and recorded in real time using the WLJY-9000 pattern chromatic color spermatozoa quality detection system.</p><p><b>RESULTS</b>Compared with parallel control group, the exposure of adult male fresh semen to a 50-Hz EMF at 0.4 mT for 15 min or 60 min could decrease significantly the motoricity (spermatozoa with a + b lever) and the activity ratio (spermatozoa with a + b + c lever)(P < 0.01). However, there were no significant differences of motoricity and the activity ratio between exposure group and control group (P > 0.05), and after exposure to a 50-Hz. EMF for 30 min the motoricity and the activity ratio of exposure group were inhibited, as compared with control group (P < 0.01 or P < 0.05). The pH value of the semen was not obvious changed (P > 0.05) when semen was exposed to a 50-Hz EMF of 0.4 mT for 15, 30 and 60 min.</p><p><b>CONCLUSION</b>In present experiment, it is suggested that the exposure of adult male fresh semen to a 50-Hz EMF in vitro could inhibit the motoricity and the activity ratio, but not affect the pH value of the semen within 60 min.</p>

Evaluation study on urine metabolomics in yinhuang rat model induced by triplet factors of rhubarb, ethanol, and α-nephthylisothiolyanate / 中国结合医学杂志

<p><b>OBJECTIVE</b>To ascertain the biomarkers capable to characterize the animal composite model of Chinese medicine (CM) yinhuang syndrome induced by triplet factors of rhubarb, ethanol, and α-nephthylisothiolyanate (abbreviated as R, E, and A below) through metabolomic study and to evaluate the established model by means of studying the sources of markers based on the changes of metabolites produced from various combinations of the three modeling drugs.</p><p><b>METHODS</b>Eighty Wistar rats allocated equally in eight groups (A-H) were treated with saline, R+E+A, R, E, A, R+E, R+A, and E+A, respectively. Rats' 12 h urine in the 14 successive experimental days were collected separately using metabolic cages and analyzed by ultra performance liquid chromatograph/time of flight mass spectrometer (UPLC/TOF-MS) to create the metabolic contour graph of urine in different groups for identifying the differences between them. The similarities and differences of metabolic network among various groups were represented from microcosmic viewpoint by pattern recognition method (principal component analysis).</p><p><b>RESULTS</b>Controlled by group A, the landing points in principal component map of various groups were apparently assorted, especially obvious on the 14th day; 19 biomarkers, which capable to represent the genesis and development process of the yinhuang syndrome in the triplet factors-induced rat model, were identified.</p><p><b>CONCLUSION</b>Metabolomic method is successfully used in evaluating the animal model of CM syndrome. Furthermore, according to the holistic view and substance changes in vivo, the influences of disease on organism were comprehensively analyzed, and the pathogenic mechanism of CM yinhuang syndrome was explored at the level of metabolomics in vivo as well.</p>

A preliminary study on role of acid sphingomyelinase in receptor clustering induced by 50-Hz magnetic fields / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the relationship among a 50-Hz MF-induced epidermal growth factor receptor (EGFR) clustering, acid sphingomyelinase (A-SMase) and ceramide (CER), and to explore the possible mechanism of receptor clustering.</p><p><b>METHODS</b>Human amnion (FL) cells were exposed to a 50-Hz sinusoidal magnetic field at 0.4 mT for 15 min with or without imipramine, a specific inhibitor of A-SMase and ceramide pretreatment. EGF treatment served as the positive control and DMSO treatment served as the solvent control. The EGFR was labeled with polyclonal anti-EGFR antibody and the clustering of EGFR was analyzed using immunofluorescence and confocal microscopy. The percentage of cells with EGFR clustering was counted and compared.</p><p><b>RESULTS</b>Both EGF treatment and 50-Hz MF exposure could induce EGFR clustering. However, the effect could be eliminated by imipramine pretreatment for 4 hours. When FL cells were incubated with ceramide following the imipramine pretreatment for 30 min, EGFR clustering induced by 50-Hz MF exposure could be recovered.</p><p><b>CONCLUSION</b>EGFR clustering induced by 50-Hz MF depends on A-SMase activity, and ceramide, as the hydrolyzate from A-SMase might participate in the process of EGFR clustering.</p>

Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride / 中国结合医学杂志

<p><b>OBJECTIVE</b>To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine.</p><p><b>METHODS</b>The liver injury in the model rat was induced by alpha-naphthylisothiocyanate (ANIT) and carbon tetrachloride (CCl(4) ) respectively, and the effects of Yinchenhao Decoction (, YCHD), a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic, on the two liver injury models were evaluated by analyzing the serum level of alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP), malondialchehyche (MDA), total bilirubin (T-BIL), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) as well as the ratio of liver weight to body weight. The experimental data were analyzed by principal component analytical method of pattern recognition.</p><p><b>RESULTS</b>The ratio of liver weight to body weight was significantly elevated in the ANIT and CCl(4) groups when compared with that in the normal control (P<0.01). The contents of ALT and T-BIL were significantly higher in the ANIT group than in the normal control (P<0.05,P<0.01), and the levels of AST, ALT and ALP were significantly elevated in CCl(4) group relative to those in the normal control P<0.01). In the YCHD group, the increase in AST, ALT and ALP levels was significantly reduced (P<0.05, P<0.01), but with no significant increase in serum T-BIL. In the CCl(4) intoxicated group, the MDA content was significantly increased and SOD, GSH-PX activities decreased significantly compared with those in the normal control group, respectively (P<0.01). The increase in MDA induced by CCl(4) was significantly reduced by YCHD P<0.05).</p><p><b>CONCLUSION</b>YCHD showed significant effects on preventing liver injury progression induced by CCl(4), and the closest or most suitable animal model for damp-heat jaundice syndrome may be the one induced by CCl(4).</p>

Preliminary study on role of lipid rafts in receptor clustering induced by 50 Hz magnetic fields and its mechanism / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the relationship among a 50 Hz magnetic field (MF)-induced epidermal growth factor receptor (EGFR) clustering,lipid rafts and acid sphingomyelinase (ASM), and to explore its possible mechanism.</p><p><b>METHODS</b>Human amnion FL cells were exposed to 50 Hz, 0.4 mT MF for 15 min. EGF treatment was used as positive control. Nystatin was employed to study lipid rafts since it could disrupt lipid rafts structure.The EGF receptors, ASM and lipid rafts were labeled with polyclonal anti-EGFR antibody, anti-ASM antibody and FITC-Cholera toxin B, respectively. The images were observed by laser confocal scanning microscope.</p><p><b>RESULT</b>Both EGF treatment and 50 Hz MF exposure could induce EGFR clustering; however, nystatin pretreatment disrupted this effect. MF exposure turned ASM (labeled with Cy3) from a diffused state in the sham exposure group to a concentrated state on the cell membrane, which co-localized with lipid rafts (labeled with FITC).</p><p><b>CONCLUSION</b>The results suggest that the EGFR clustering induced by 50 Hz MF depends on intact lipid rafts on cellular membrane, and the ASM might participate in the process of EGFR clustering.</p>

Blocking 1800 MHz mobile phone radiation-induced reactive oxygen species production and DNA damage in lens epithelial cells by noise magnetic fields / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate whether the exposure to the electromagnetic noise can block reactive oxygen species (ROS) production and DNA damage of lens epithelial cells induced by 1800 MHz mobile phone radiation.</p><p><b>METHODS</b>The DCFH-DA method and comet assay were used respectively to detect the intracellular ROS and DNA damage of cultured human lens epithelial cells induced by 4 W/kg 1800 MHz mobile phone radiation or/and 2 muT electromagnetic noise for 24 h intermittently.</p><p><b>RESULT</b>1800 MHz mobile phone radiation at 4 W/kg for 24 h increased intracellular ROS and DNA damage significantly (P<0.05). However, the ROS level and DNA damage of mobile phone radiation plus noise group were not significant enhanced (P>0.05) as compared to sham exposure group.</p><p><b>CONCLUSION</b>Electromagnetic noise can block intracellular ROS production and DNA damage of human lens epithelial cells induced by 1800 MHz mobile phone radiation.</p>

Noise magnetic fields mitigates the inhibition of secretion function of primary human villous trophoblasts induced by 50 Hz magnetic fields / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore the effects of 50 Hz sinusoidal magnetic fields (MF) on secretion function of primary human villous trophoblasts in vitro, and the interference effect of "noise" MF.</p><p><b>METHODS</b>The trophoblasts were isolated from human villus by trypsin digestion and incubated in DMEM medium.Then the trophoblasts were exposed to 0.4 mT 50 Hz MF and/or "noise" MF respectively for different durations. Each exposure group was matched with one control group which was from the same villus and cultured with the same condition except the MF exposure. The concentrations of human chorionic gonadotropin (HCG) and progesterone in the culture medium were measured by immunofluorescence. Statistical significance of differences between means was determined by one way-ANOVA with P<0.05 considered significant.</p><p><b>RESULT</b>50 Hz MF inhibited the HCG and progesterone secretion significantly when exposure for 72 h (compared with control group, P<0.05). There was no significant change of HCG and progesterone secretion when trophoblasts were exposed to 0.4 mT "noise" MF within 72 h (compared with control group, P>0.05). However, by superimposing the "noise" MF, the inhibition of HCG and progesterone secretion of trophoblasts induced by 50 Hz MF was eliminated.</p><p><b>CONCLUSION</b>The exposure to 50 Hz MF for long period could inhibit trophoblasts secreting HCG and progesterone, and the "noise" MF with the same intensity could eliminate the effects induced by 50 Hz MF.</p>

Effects of 50 Hz magnetic fields exposure on secretion of primary human villous trophoblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the possible effects of 50 Hz magnetic fields (MF) exposure on HCG and progesterone secretion of human villous trophoblasts in vitro.</p><p><b>METHODS</b>The trophoblasts were isolated from human villus by trypsin digestion and incubated in DMEM medium. Then the trophoblasts were exposed to 0.2 mT, 0.4 mT 50 Hz MF for 6 h, 12 h, 24 h, 48 h and 72 h, respectively. Each exposure group was matched to one control group which was from the same villus and cultured with the same condition except the 50 Hz MF exposure. The concentration of human chorionic gonadotropin (HCG) and progesterone in the culture medium was detected by electrochemiluminescence immunoassay. Statistical significance of differences between means was determined by one way-ANOVA with P < 0.05 considered significant.</p><p><b>RESULTS</b>Exposure of trophoblasts to 50 Hz MF at 0.2 mT intensity within 72 h did not affect the secretion level of HCG and progesterone (compared with blank control, P > 0.05). There was also no significant change of the secretion level of HCG and progesterone when trophoblasts were exposed to 0.4 mT 50 Hz MF within 48 h (compared with blank control, P > 0.05). However, 50 Hz MF inhibited the HCG and progesterone secretion significantly with exposure for 72 h (compared with blank control, P < 0.05).</p><p><b>CONCLUSION</b>The exposure to 50 Hz MF for long period could inhibit trophoblasts excreting the HCG and progesterone, and the threshold intensity may be between 0.2 mT and 0.4 mT.</p>

Apoptosis-related gene expression of human villous trophoblasts exposed to 50 Hz magnetic field / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study apoptosis-related gene expression of human villous trophoblasts exposed to 50 Hz magnetic field and to investigate the possible mechanism of human reproductive health effects caused by 50 Hz magnetic field.</p><p><b>METHODS</b>Cultured human villous trophoblasts were exposed to 50 Hz magnetic field at 0.4 mT for 6, 48, 72 hours. Gene expressions of Bcl-2, Bax, Caspase-3, p53 and Fas were analyzed using real-time reverse transcription polymerase chain reaction (RT-PCR) assay.</p><p><b>RESULTS</b>Within 72 hours, the average fold change for each gene was near 1.00, and there was no significant difference on expression pattern in each gene between exposure and control groups (P > 0.05).</p><p><b>CONCLUSION</b>0.4 mT 50 Hz magnetic field does not affect the apoptosis-related gene expression of human villous trophoblasts in vitro.</p>

GSM 1,800 MHz radiofrequency electromagnetic fields induced clustering of membrane surface receptors and interference by noise magnetic fields / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the possible effect of exposure to GSM 1,800 MHz radiofrequency electromagnetic fields (RF EMF) on epidermal growth factor (EGF) receptor and its possible interference by noise magnetic fields (MF).</p><p><b>METHODS</b>Chinese hamster lung fibroblasts (CHL) were exposed to 1,800 MHz RF EMF (modulated by 217 Hz or 50 Hz, or unmodulated), 2 microT noise MF, and RF EMF combined with 2 microT noise MF for 15 min, respectively. The specific absorption rates (SARs) of RF EMF were 0.1, 0.5, 1.0, 2.0 and 4.0 W/kg. Commercial EGF (1 ng/ml) treatment was used as positive control. EGF receptors on the cell membrane were observed under a laser scanning confocal microscope after indirect immunofluorescence staining.</p><p><b>RESULTS</b>EGF receptor clustering was induced after exposure to GSM 1,800 MHz RF EMF modulated by 217 Hz or 50 Hz MF at SARs of 0.5, 1.0, 2.0, 4.0 W/kg for 15 min as induced by 1 ng/ml EGF, but not at SAR of 0.1 W/kg. And no EGF receptor clustering was found in cells after exposure to unmodulated RF EMF or 2 microT noise MF. In addition, superposition of 2 microT noise MF could inhibit the EGF receptor clustering induced by GSM 1,800 MHz RF EMF.</p><p><b>CONCLUSION</b>EGF receptor clustering in CHL cells can be induced by GSM 1,800 MHz RF EMF at the lowest SAR of 0.5 W/kg and inhibited by noise MF. The modulation of wave may play an important role in the inducement of receptor clustering after RF exposure.</p>

Effect of geniposide on serum IL-1beta and TNF-alpha of rheumatoid arthritis rats / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of geniposide on serum IL-1beta and TNF-alpha levels of rheumatoid arthritis rats, as well as the mechanism of this drug.</p><p><b>METHOD</b>To establish an experimental rat model of type II collagen-induced arthritic (CIA). The inhibitory effects on paw edema were observed, and serum IL-1beta and TNF-alpha levels were determined in experimental rats.</p><p><b>RESULT</b>Compared with the model, geniposide delayed the starting time of right paw edema significantly, and the levels of serum IL-1beta and TNF-alpha were significantly decreased by geniposide at high dose or medium dose (P < 0.01).</p><p><b>CONCLUSION</b>Geniposide can lower serum IL-1beta and TNF-alpha levels in rheumatoid arthritis rats. The effect may be close related to inhibitory development of rheumatoid arthritis by the agent.</p>

Study on the collecting method of constituents absorbed into the blood from anti-V capsule / 中国中药杂志

<p><b>OBJECTIVE</b>Aim To establish the condition and parameters for collecting the effective constituents of Anti-V capsule.</p><p><b>METHOD</b>Based on HPLC, using chlorogenic acid and baicalin which are the main constituents absorbed in the blood as the marker substance, the best technical process of macropore absorbed resin was evaluated by the quantity of two compounds in the collected part.</p><p><b>RESULT</b>After the abstraction of the capsule, the collection method of NKA-2 macropore absorbed resin as sorbent and 5 bed volume 50% ethanol as mobile phase was definited as the best craftwork. In the collected part, the content of chlorogenic acid and baicalin was five times than in the capsule.</p><p><b>CONCLUSION</b>In guide of serum pharmacochemistry, macropore absorbed resin is the effective method to collect the constituents absorbed in blood of Anti-V capsule. At the same time, this method provides a way to research other Compound medicine.</p>

Superposition of noise magnetic fields inhibits clustering of fibroblast membrane surface receptors induced by 50 Hz magnetic fields in Chinese hamster lungs / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the possible induction effect of exposure to 50 Hz magnetic field (MF) on clustering of cell membrane surface receptors for epidermal growth factor (EGF) and tumor necrosis factor (TNF), the starting site of signals of biological effects, and its possible intervention effect.</p><p><b>METHODS</b>Lung fibroblasts of Chinese hamster (CHL) were exposed to EGF, TNF, 0.4 mT 50 Hz MF, 0.4 mT noise MF, and 0.4 mT 50 Hz MF combined with 0.4 mT noise MF. Respectively, for different durations, following the treatment, EGF and TNF receptors on the cell membrane were marked by corresponding antibodies with immunohistochemical method, then observed under a confocal microscope.</p><p><b>RESULTS</b>Clustering of cell membrane receptors could be induced 5 min after treatment with EGF and TNF, as well as with 50 Hz MF at 0.4 mT, which reached the peak in 15 min. While noise MF with the same intensity did not induce clustering of cell membrane receptors. Superposition of noise MF with the same intensity could inhibit clustering of cell membrane receptors induced by 50 Hz MF.</p><p><b>CONCLUSION</b>Clustering of EGF and TNF receptors on the cell membrane could be induced by 50 Hz MF, suggesting that membrane receptors would be one of the sites where MF signals coupled, and noise MF with the same intensity could inhibit these effects.</p>

Optimization of Conditions on Submerged Fermentation of Bacillus cereus / 微生物学通报

The fermentation of Bacillus cereus DLSL-2 was investigated through single-factor test. The optimized fermentation conditions are: temperature 30℃,initial pH 7.0,250 r/min. Uniform design was used in shaking flask to optimize the fermentation medium of bacillus cereus . The most suitable medium was identified as follows:4.88% defated soybean power ,1.45% maize starch, 0.106% K 2HPO 4, 1.78% yeasts extract, 5.6% inoculum. the optimized medium allowed the B.cereus biomass concentration to be increased from 3.2?109 cfu/mL to 7.1?109 cfu/mL.

Studies on the metabonomics of rat liver injury induced by ethanol and interfering effects of Yin Chen Hao Tang / 中国药理学通报

Aim To determine potential biomarkers contributed to occurrence, development and recovery of ethanol-induced liver injury in rat and elucidate hepatoprotective effect of Yin Chen Hao Tang based on metabonomic investigation. Methods A UPLC-Q-TOF/MS based metabonomic method was developed for investigating trajectory change and inter-relationship of urinary metabolome of rats with different treatments. Results Four potential biomarkers were determined which contributed to occurrence, development and recovery of ethanol-induced liver injury in rat, and Yin Chen Hao Tang could significantly recover trajectory change in disorder. Conclusion The developed method was successfully applied to investigate ethanol-induced liver injury in rat, and also hepatoprotective effect of Yin Chen Hao Tang was elucidated.