Objective To investigate the relationship between nitric oxide and contractile disfunction of junctional zone of smooth muscle by detecting the expression of nitric oxide in uterine junctional zone in patients with adenomyosis.Methods From February 2012 to July 2012,23 patients with adenomyosis undergoing hysterectomy as adenomyosis group matched as 16 patients with cervical carcinoma or ovarian cancer were enrolled as control group in Center of Minimally Invasive Gynecology,Beijing Obstetrics and Gynecology Hospital.Location of nitric oxide synthase (inducible NOS and endothelial NOS) in uterine junctional zone was analyzed by immunohistochemistry.Smooth muscle cells of junctional zone were cultured and cellular mRNA levels of nitric oxide synthase were detected by real-time polymerase chain reaction.Furthermore,concentration of nitrites in lysates of fresh junctional zone tissues and cultured cells were determined by Griess reaction.Results (1) The expression of nitric oxide synthase proteins in smooth muscle cells of junctional zone tissues:the staining of iNOS was intensive in smooth muscle cells of junctional zone in patients with adenomyosis,while staining of eNOS was scant.Both staining of iNOS and eNOS were not observed in control group.(2) The expression of NOS genes in cultured smooth muscle cells from adenomyosis:mRNA level of iNOS was elevated by 1.5 times (P ＜0.01) while eNOS level was reduced by 2.6 times when compared with those in control groups (P ＜ 0.05).(3) Nitrites concentration in lysates of junctional zone tissues and cultured cells:concentrations of nitrites in lysates of 100 mg tissues were respectively (37 ± 8) μmol/L and (28 ± 5) μmol/L in adenomyosis and control group (P ＞ 0.05).While concentrations of nitrites in 1 × 106 cells were respectively (11.8 ± 0.6) μmol/L and (10.8 ± 1.5) μmol/Lin adenomyosis and control group,which did not reach statistical difference (P ＞ 0.05).Conclusion Upregulation of nitric oxide might be associated with contractile disfunction of smooth muscle cells in uterine junctional zone of patients with adenomyosis.

72 cases of treatments on insomnia were recorded in the medical books of Ming and Qing dynasties(A.D.1368～1911 years).The compatibility regularity of treating insomnia in these 72 cases were statistically analyzed with frequency and the R type cluster analysis methods. Combining the data and modern recognition, compatibility and medication regularity of treating insomnia was further analyzed.

A suitable ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of 11 mycotoxins with isotope internal standard in malt. The mycotoxins in malt were extracted and purified by one-step ultrasonic extraction procedure using acetonitrile/water/acetic acid (80 : 19 : 1), and then detected and confirmed by UPLC-MS/MS, and quantified by isotope labeled AFB1 ([13C17]-AFB1) and ZEN ([13C18]-ZEN) internal standards. Rapid separation of the 11 mycotoxins was successfully achieved on a Phenomenex Kinetex C18 column (100 mm x 2.1 mm, 2.6 μm) with gradient elution using the mobile phase of methanol containing 0.1% formic acid and 2 mmol x L(-1) ammonium acetate in water. Simultaneous acquisition was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) source operated in both positive and negative ionization modes. The established method provided a good linearity for the 11 mycotoxins within their respective linear ranges with correlation coefficients all higher than 0.999 1. The average recoveries ranged from 75.0% to 117.0% with relative standard deviations (RSDs) below 5.1%. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.05 to 30 μg x kg(-1) and 0.15 to 87.5 μg x kg(-1), respectively, which were below the maximum residue levels (MRLs) set by the European Union. Twenty malt samples were analyzed and nine samples were detected with mycotoxins, which were confirmed according to the same fragment ions found in positive samples and the standards at the same retention time. This study has demonstrated that the one-step extraction procedure of mycotoxins from complex matrices coupled to UPLC-MS/MS method is simple, quick, accurate and sensitive for quantitative and qualitative analysis of multiple mycotoxins in malt.
Chromatography, High Pressure Liquid ; Fermentation ; Hordeum ; chemistry ; Limit of Detection ; Mycotoxins ; analysis ; isolation & purification ; Tandem Mass Spectrometry

Objective To observe the effects of long term injection sodium salicylate on the auditory brain-stem response(ABR)and expression of glutamic acid decarboxylase -67(GAD67) in rat inferior colliculus .Methods Eighteen healthy Wistar rats were randomly divided into three groups :the sodium salicylate group (intramuscular injection of 10% sodium salicylate ,175 mg/kg ,twice daliy for 28 days) ,the saline group (intramuscular injection with saline on same does at the same time) ,the control group (without any treatment) .The rats received ABR after modeling ,then were decapitated and inferior colliculus tissues were stripped .Western blot was used to study the dif-ferent expression of GAD67 protein levels in the three groups .Results Compared with the saline group and control group ,ABR thresholds of the sodium salicylate group were significantly elevated and latency of wave Ⅲ was aslo sig-nificantly prolonged(P<0 .01) ,while there was no significant difference between the saline group and the control group(P>0 .05) .The inferior colliculus GAD67 protein expression level of sodium salicylate group was significantly higher than the saline group and control group(P<0 .01) ,while there was no significant difference between the saline group and the control group(P>0 .05) .Conclusion Long term injection of sodium salicylate can cause a change in the inferior colliculus of GAD67 protein expression and the up regulation of GAD67 expression may occur as a com-pensatory response to increase inhibiting effect .The change of GAD67 protein expression is likely as a compensatory and regulatory mechanisms for sodium salicylate ototoxicity .

Objective To investigate the expression of RhoA and Rho kinase in junctional zone (JZ) of adenomyosis and normal myometrium and explore its relationship with severity of dysmenorrheal.Methods From Mar.to Dec.2012,32 cases with adenomyosis undergoing hysterectomy were enrolled as adenomyosis group including 18 cases with proliferative endometrium and 14 cases with secretory endometrium matched with 29 cases with hysterectomy due to cervical disease and ovarian tumor as control group including 12 cases with proliferative endometrium and 17 cases with secretory endometrium in Beijing Obstetrics and Gynecology Hospital Affiliated to Capital Medical University.JZ smooth muscle cells were isolated and cultured immediately after the operation.The expression of mRNA and protein of RhoA and ROCK Ⅰ in JZ in two groups were measured by real-time fluorescence quantitative RT-PCR and western blot.Results (1) The mRNA expression of RhoA and ROCK Ⅰ in JZ of adenomyosis group did not show cyclic change.In proliferative phase,the expression of RhoA and ROCK Ⅰ (1.41 ±0.16,1.05 ±0.15) was not significantly higher than that in secretory phase (1.17 ± 0.25,0.98 ± 0.10) (P ＞ 0.05).While JZ in control group,it showed obviously cyclic change.The expression level of them in proliferative phase(0.93 ±0.10,1.00 ± 0.18) was significantly higher than that in secretory phase (0.48 ± 0.03,0.55 ± 0.05) (P ＜0.05) ; It also showed that expressions of RhoA and ROCK Ⅰ in adenomysis group were significant higher than those in the control (P ＜ 0.05).(2) The mRNA and protein expression of RhoA and ROCK Ⅰ was positively correlated in each of two groups (r =0.48,P ＜ 0.01 ; r =0.67,P ＜ 0.01).(3) The expression of RhoA and ROCK Ⅰ were 1.66 ±0.19,1.32 ±0.11 in severe dysmenorrheal,1.28 ±0.12,1.09 ±0.08 in moderate dysmenorrheal and 0.93 ± 0.09,0.81 ± 0.06 in mild dysmenorrheal,it all reached statistical difference when compared the other group.(All P ＜ 0.05).Conclusions The expressions of RhoA and ROCK Ⅰ in JZ in adenomyosis group were higher than those in control group,and positively correlated with the severity of dysmenorrheal in adenomysis group,but it does not change with the menstrual cycle.High expression of RhoA and ROCK Ⅰ might be involved in abnormal contraction of uterine myometrium and correlated with the dysmenorrheal in adenomysis.

Objective To estimate the expression of ER and PR in the endometrium of both intrauterine adhesions (IUA) and non-IUA specimens. Methods The endometrium specimens from patients undergoing hysteroscopy for confirmed moderate IUA (n=20: 10 in proliferative phase, and 10 in secretory phase) were enrolled as the IUA group in Beijing Obstetrics and Gynecology Hospital from October 2014 to August 2015. The specimens scheduled for hysteroscopy due to infertility were recruited into the control group (n=26: 13 in proliferative phase, and 13 in secretory phase). Immunohistochemistry and quantificational real-time PCR (qRT-PCR) were used to detect the expression of ER-α, ER-β and PR in endometrium with different menstrual period in both groups. Results (1) Location: in both groups, the expression of ER-α, ER-β and PR appeared in the endometrial glandular epithelial cells and the stromal cells of the endometrium. The positive brown granules of ER-α, ER-β and PR appeared mainly in cell nucleus. (2) ER-α and ER-β in the endometrium:the protein expression of ER-α and ER-β in IUA group (proliferative phase: 0.657 ± 0.028, 0.493 ± 0.023; secretory phase: 0.537 ± 0.020, 0.365 ± 0.031) were significantly higher than those of control group (proliferative phase: 0.586 ± 0.025, 0.437 ± 0.022; secretory phase:0.459 ± 0.025, 0.323 ± 0.017;all P<0.01). And the ER-αand ER-βmRNA expressions in IUA group were 2.524 ± 0.296, 1.947 ± 0.339, higher than those of control group in the proliferative phase (all P<0.01), and in the secretory phase (1.977±0.333, 1.345±0.292) were also higher than those in the control group (all P<0.01). (3) PR in the endometrium: the protein expression of PR was not significantly different between IUA group (proliferative phase:0.248±0.025, secretory phase:0.194±0.024) and control group (proliferative phase: 0.234 ± 0.019, secretory phase: 0.186 ± 0.020; P=0.162, 0.359). Meanwhile, there were no statistical differences in the mRNA expression of PR in both groups with different menstrual period (proliferative phase: 1.144 ± 0.384 versus 0.981 ± 0.306, secretory phase: 0.763 ± 0.237 versus 0.631 ± 0.203; P=0.270, 0.166). (4) ER and PR expression in menstrual cycles: the expression of ER-α, ER-β and PR in the IUA group changed with the menstrual cycles, and their expression in the proliferative phase were higher than those in the secretory phase (all P<0.05). Conclusions The expression of ER-α and ER-β in the endometrium of IUA patients changes with menstrual cycle, and are higher compared with those in normal endometrium. No difference is found in the PR expression between the two groups.

Objective To study changes of auditory brainstem evoked potentials (ABR)and the expression of glial fibrillary acidic protein (GFAP)during the critical period of the development of the auditory cortex in rats. Methods Fifty Sprague - Dawley (SD)rat pups were divided into five groups(n=10 per group):postnatal 14 days group,postnatal 21 days group,postnatal 28 days group,postnatal 35 days group and postnatal 42 days group.The rats were decapitated after the evaluation of ABR.The auditory cortex was taken and immunohisto-chemical staining was used to detect the expression of GFAP in rat auditory cortex in each group.The protein ex-pressions of GFAP in rat auditory cortex in each group were tested by western blot methods respectively.ResuIts The ABR thresholds gradually decreased along with the increasing days after the birth.The average ABR thresholds in postnatal 14 days group,postnatal 21 days group,postnatal 28 days group,postnatal 35 days group and postnatal 42 days group were (84.5 ± 4.97)dB SPL ,(70.5±3.69)dB SPL ,(58.5±5.80)dB SPL,(37.0±4.83)dB SPL and(35.5±3.69)dB SPL,respectively.There were no significant differences in ABR thresholds between the postnatal 35 days group and the postnatal 42 days group (P>0.05),whereas,significant differences were found among the other groups(P<0.05 or P<0.01.GFAP expression in auditory cortex gradually increased in all five groups.The average integrated optical density (IOD)of postnatal 14 days group,postnatal 21 days group,postnatal 28 days group,postnatal 35 days group and postnatal 42 days group were 474.36±234.56,1465.93 ±474.96,2163.06 ± 353.36,6572.01±808.88 and 7244.37±932.90,respectively.The differences among all five groups were statisti-cally significant (P<0.05 orP<0.01 ).The protein levels of GFAP in the auditory cortex of postnatal 14 days group,postnatal 21 days group,postnatal 28 days group,postnatal 35 days group and postnatal 42 days group were 1.00±0.06,3.07±0.07,4.92±0.05,6.88±0.03 and 8.92±0.04.The differences among all five groups were sta-tistically significant (P<0.05).ConcIusion The ABR threshold gradually decreased and GFAP expression in audi-tory cortex gradually increased from postnatal 14 days to postnatal 42 days ,suggesting that astrocytes may promote the development and maturation of the auditory cortex and the auditory central system in rats.

Objective To study the expression of TrkB and c-fos in rat auditory cortex after long -term ad‐ministration of sodium salicylate and the mechanisms of salicylate ototoxiclty .Methods Normal adult rats were di‐vided into three groups :normal group without any treatment ,long -term treatment group given i .m .injections , 175 mg/kg ,twice daily at 9:00 am and 6 :00 pm for consecutive 14 days and the recovered group using the same method as the chronic group except that they were sacrificed 28 days after salicylate treatment ceased .After the de‐tection of ABR ,rats were sacrificed after deep anesthesia ,and the auditory cortexes were dissected rapidly .Real-time PCR and Western-blot were explored to detect the expression of TrkB and c-fos .Results The average ABR threshold of normal rats was 36 ± 2 .23 dB SPL .The average ABR threshold of long -term group was 41 .3 ± 3 .31 dB SPL ,which was significantly increased than normal group(P<0 .01) .The average ABR threshold of the recoveredgroup was 38 .6 ± 5 .51 dB SPL .There were no differences between the recovered group and normal group(P>0 . 05) .The expression of c-fos mRNA(1 .24 ± 0 .09)and protein(0 .70 ± 0 .12)in the long -term group were signifi‐cantly increased ,when compared with the normal group .The c-fos mRNA(1 .23 ± 0 .04)and protein(0 .68 ± 0 .08) expression in the recovered group were also significantly increased compared with the normal group .The expression of TrkB mRNA(1 .26 ± 0 .10)and protein(1 .85 ± 0 .17)in the long -term group were significantly increased ,when compared with the normal group .The TrkB mRNA(1 .23 ± 0 .07)and protein(1 .80 ± 0 .08)expression in the recov‐ered group were also significantly increased compared with the normal group .Conclusion The inereased expression of c-fos in the long -term group may be related to the enhancement of central auditory function .The inereased ex‐pression of Trk-B in the long -term group might be involved in instability of synaptic plasticity in tinnitus .

Objective Discuss the clinical efficacy of caffeine citrate and aminophylline in treatment of primary apnea of prematurity.Methods 80 cases of primary apnea of prematurity were selected.They were divided into two groups randomly.The control group (40 cases) received aminophylline treatment and the observation group (40 cases) received caffeine citrate therapy.The efficacy of caffeine citrate and aminophylline in treatment of primary apnea ofprematudty was evaluated by efficacy,primary apnea episodes and disappeared time,adverse reaction during treatment.Results The effective rate of observation group was 85％,the effective rate of control group was 75％.The observation group had higher efficiency (P ＜ 0.05).The frequency of apnea of observation group was less than that of the control group.The disappearance time of observation group was shorter than that of the control group (P ＜ 0.05).During the treatment,the tachycardia,feeding intolerance,bronchopulmonary dysplasia and other adverse reactions rate of observation group was lower than that of the control group (P ＜ 0.05).Conclusion Compared with aminophylline,caffeine citrate had a good therapeutic effect of primary apnea of prematurity.It can reduce apnea frequency,eliminate clinical symptoms with high safety.It was an ideal drug for treatment of primary apnea.

To study the effect of Gegen Qinlian decoction and its major effective components on five hepatic microsomal CYP450 isozymes in rats. The in vitro hepatic microsomal incubation technique was used to co-culture Gegen Qinlian decoction and its major effective components together with each probe substrate. HPLC-MS/MS was used to establish the analytical method for metabolites of the five isoform probe substrates of CYP450 isozymes, detect the linearity among micoromal protein concentration, incubation time and metabolite formation amount. And HPLC-MS/MS was applied to determine the formation rate (V) of corresponding metabolites (acetaminophen, 4-OH-chlorzoxazone, dextrophan, 6-OH-chlorzoxazone and 6β-hydroxytestosterone) specific probe substrates of the five isoform probe substrates of CYP450 isozymes (phenacetin, polbutamide, dextromethorphan, chlorzoxazone, testosterone), in order to determine the activity of each isozyme. The result showed good linearity among acetaminophen, 4-OH-tolbutamide, dextrophan, 6-OH-chlorzoxazone and 6β-hydroxytestosterone, satisfactory precision, stability and average recovery, suggesting the method was feasible. The optimized in vitro microsomal incubation conditions conformed to the requirements in the guideline of drug-drug interaction. Gegen Qinlian decoction showed different degrees of inhibitor effect on 5 CYP450 isoforms (CYP1A2, CYP2C11, CYP2D2, CYP2E1, CYP3A1/2). Its major effective component berberine could inhibit each CYP450 isoform at high concentrations (except for CYP1A2, CYP3A1/2).
Animals ; Chromatography, High Pressure Liquid ; methods ; Cytochrome P-450 Enzyme Inhibitors ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Isoenzymes ; antagonists & inhibitors ; Liver ; enzymology ; Rats ; Tandem Mass Spectrometry ; methods