A sensitive and selective liquid chromatography–tandem mass spectrometric (LC ? MS/MS) method was established to determine 2-oxo-clopidogrel, a crucial intermediate metabolite in human plasma. A chromatographic separation was performed on a Sapphire C18 column following a liquid–liquid extraction sample preparation with methyl t-butyl ether. Detection was carried out on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) with an electrospray ionization (ESI) mode. The method was validated in terms of specificity, accuracy, precision and limit of quantification. The calibration curves ranged from 0.50 to 50.0 ng/mL with good linearity. The stability was fully validated with addition of 1,4-dithio-DL-threitol (DTT) into the plasma sample prior to and in the preparation procedure. The validated method was proved to be suitable for use in pharmacokinetic study after single oral administration of 75 mg clopidogrel tablets in human subjects, which could make contribution to intensive study of the clinical drug–drug interactions of clopidogrel and individual treatment.

Objective To identify the role of resistin in insulin resistance(IR) by endoplasmic reticulum(ER) stress in human umbilical vein endothelial cells ( HUVECs) and in rats.Methods HUVECs were cultured in vitro and disposed by resistin (R) or tauro ursodesoxycholic acid (tudca).Expressions of GRP78, P-akt and P-eNOS were determined using Western blotting.Thoracic aortic rings were made and their dilation function exposed to different concentrations of insulin was detected.Changes of vascular morphology were observed by HE staining.Results Results of Western blotting showed that expression of GRP78 was remarkably increased,but P-akt and P-eNOS were markedly decreased in R group.However, there was no difference in expressions of GRP78, P-akt and P-eNOS between tudca group and control group.The insulin induced vasodilation was decreased in R group and there was no difference between tudca group and control.Using HE staining, the R group showed significant medial thickening and proliferation of smooth muscle.Conclusion Resistin can induce insulin resistance in vascular endothelium cells by ER stress.

ABSTRACT:Objective To investigate whether medial cerebellar nucleus may be involved in the pathogenesis of post-stroke depression (PSD)and explore the pathway that mediates this effect.Methods Healthy SD rats were randomly divided into five groups:control group,stroke group,PSD group,medial cerebellar nucleus (Med)lesion group,and superior cerebellar peduncle (xscp)lesion group.The ethological score of depression was evaluated.The neurotransmitters of Glu and GABA in the lateral hypothalamic area were detected by high performance liquid chromatography.Results Compared with those in control group,the levels of GABA and Glu expressions did not significantly differ in the lateral hypothalamic area in stroke group (P > 0.05 ).In contrast,the levels in PSD group,Med lesion group and xscp lesion group were lower than those in stroke group to different extent (P <0.01). Conclusion These results preliminarily show that medial cerebellar nucleus may participate in the pathogenesis of post-stroke depression,which may be mediated by the cerebellum-hypothalamic pathway.

Objective To screen the effective silencing targets of P21 gene at the cellular level in rhesus monkey . Methods To detect the expression of P21 gene in COS-7 cells ( derived from the kidney of African green monkey , Cerco-pithecus aethiops).Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and in-serted into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into COS-7 cells respective-ly.The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by West-ern blot assay .Results Four gene-silencing sequences were screened that lied in 541-561 bp, 542-562 bp, 215-239 bp, and 624-648 bp of the rhesus monkey P21 mRNA.Their silencing rate was (91.82 ±3.21)%, (82.47 ±2.48)%, (81.31 ±2.69 )% and ( 87.35 ±4.59 )%, and the protein expression was ( 11.97 ±0.70 )%, ( 20.22 ±0.65 )%, ( 23.21 ± 0.63)%and (14.42 ±0.86)%, respectively.Conclusions Four effective silencing target sequences are screened at cel-lular level , which can be used in gene silencing research of rhesus monkeys .

Objective To study the inclusion mechanism of 14-deoxyandrographolide and ?-cyclodextrin in inclusion ratio,molecular interaction between host and guest,inclusion part and stability of inclusion compound.Methods The formation of inclusion compound was confirmed by dfferential thermal analysis(DTA);The equilibrium constants of inclusion compound were determined and the thermodynamic constants were calculated by UV;Inclusion parts were confirmed by IR and 1H-NMR.Results Ka=1 292.631 L/mol;?G=-16.854 8;?H=-25.532;?S=-0.029 12.It was clearly that all five lactone ring and decalin ring of 14-deoxyandrographolide in indusion compound changed in IR and 1H-NMR.Conclusion Inclusion host-guest ratio is 1∶1,Van der Waals forces play a major role in the process.Inclusion site is decalin ring and five lactone ring.The inclusion compound is more stable under normal temperature.

Objective To investigate the effects of ursolic acid (UA) on cisplatin (CDDP)-induced expres_sion of transient receptor potential vanilloid 1 (TRPV1) in mouse cochlea .Methods Sixty BALB/c mice were ran_domly divided into 4 groups (15 mice in each group) and received introperitoneal injection once daily for 5 days:Control group (normol saline) ,UA group (80 mg/kg/day) ,CDDP group (4 .5 mg/kg/day) ,and CDPP (4 .5 mg/kg/day) plus UA group (80 mg/kg/day) .The expression of TRPV1 in mouse cochlea was determined by immuno_histochemistry ,microscope image analysis and western blot ,and auditory thresholds were evaluated by auditory brainstem response (ABR) measurement .ResuIts The expression of cochlea TRPV1 and ABR threshold shift was significantly increased in the mice treated with CDDP (P< 0 .05) ,as compared with control mice .These effects were prevented by UA treatment (all P<0 .05) .Furthermore ,a linear relationship analysis revealed that the ex_pression of cochlea TRPV1 was significantly correlated with ABR threshold shift(|r|>0 .7 , P<0 .05) .ConcIusion UA effectively attenuated CDDP -induced ototoxicity and improved auditory function through inhibition of TR_PV1 .

Objective To explore the mechanism of the low-frequency electrical stimulate on pedunculopontine nu-cleus to treat the Parkinson (PD) through observinge the low-frequency electrical stimulation of Pedunculopontine Nucleus (PPN) in PD rat model and the effects of neurotransmitters (GPi) neurons discharge in the medial part of the globus pallidus. Methods Thirty SD rats were randomly assigned to the control group and the PD model group, with 15 in each group. PD model was established through injecting 6-OHDA into Substantia nigra compact (SNc) of black rat. Effect of low frequency electrical stimulation, micro-electrophoresis glutamate (Glu) and its receptor blocking breaking agent MK-801,γ-aminobu-tyric acid (GABA) and its receptor antagonist bicuculline (BIC) on discharge of rat neuron GPi was examined using extracel-lular unit recording methods through seven glass microelectrode recording. Results When stimulated by low frequency electrical stimulation of PPN, reactions from the control group and neuronal response GPi in PD rats were inhibited. The aver-age discharge frequency was reduced compared to pre-stimulation (P < 0.01). Micro-electrophoresis and BIC Glu excite neurons while microiontophoresis MK-801 and GABA restrain neurons. In the background of micro-electrophoresis BIC’s excitatory effects on neuron, low-frequency electrical stimulation on PPN reduced neuronal firing frequency. And in the background of inhibition effect of micro-electrophoresis MK-801, low-frequency stimulation PPN further restrain neuronal discharge frequency. Conclusion Low frequency electrical stimulation inhibits GPi PPN neuronal activity probably though regulating neurons projecting to the Glu and GABA nerve pathways in GPi neuron.

Objective To study the effect of high frequency stimulation (HFS) in pedunculopontine nucleus (PPN) on the neuronal activities of globus pallidus internus (Gpi) in Parkinson’s disease (PD) model rats, and the mechanisms there-of. Methods Seventy male Sprague-Dawley rats were divided into two groups, control group (n=30) and PD model group (n=40). PD rat model was established by the injection of 6-OHDA into substantia nigra pars compacta (SNc) on the right side of the brain with stereotactic technique. Electrophysiological recordings were made in anaesthetized rats to investigate the ef-fects of HFS-PPN on the firing rate of the GPi neurons. Brain microdialysis combined with high-performance liquid chroma-tography was applied to detect glutamate (Glu) andγ-aminobutyric acid (GABA) levels in GPi. Results HFS-PPN caused an excitatory reaction of the majority of neurons recorded in the GPi in PD model group and control group. The mean firing rate of GPi excited neurons was significantly increased (P﹤0.01). The levels of Glu were reduced under HFS-PPN and the levels of GABA were not affected (P>0.05).Conclusion HFS-PPN heightened the electrical activity of GPi neurons and re-duced the level of Glu. These excitatory effects were probably realized by PPN-GPi direct path or other indirect path.

OBJECTIVE:To observe the effect of atorvastatin combined with methylprednisolone on the liver function of ne-phrotic syndrome patients. METHODS:The data of 93 patients with primary nephritic syndrome were retrospectively analyzed and divided into atorvastatin group,methylprednisolone group and combination group by different medication. Atorvastatin group was orally given atorvastatin 20 mg at bedtime,once a day+aspirin;methylprednisolone group was orally given methylprednisolone 0.8 mg/kg in the early morning,once a day+aspirin;combination group was given atorvastatin+methylprednisolone+aspirin(the same usage and dosage with the above-mentioned groups). The course was 4 weeks. The clinic data was observed,including ALT,AST, GGT,TB and DB before and after treatment,the incidence of patients with drug-induced liver disease and prognosis of patients with drug-induced liver disease. RESULTS:After treatment,the ALT,AST and GGT in atorvastatin group and combination group were significantly higher than before,with significant difference(P<0.05);compared with other parameters and all indexes in methylprednisolone group before and after treatment,there were no significant differences(P>0.05). There was no significant dif-ference in the elevated rate of ALT among groups(P>0.05);the incidence of drug-induced liver disease in combination group was significantly higher than atorvastatin group and methylprednisolone group,with significant difference(P<0.05). ALT in combina-tion group was significantly decreased and returned to pretreatment levels after atorvastatin withdrawal and 2 weeks of hepatoprotec-tants treatment for 7 patients with drug-induced liver disease. CONCLUSIONS:Atorvastatin combined with methylprednisolone has high risk on liver function in the treatment of nephrotic syndrome. Pretreatment levels can be recovered by both drug withdrawal and symptomatic treatment.

Objective To explore a set of suitable content and methods of evaluation for nursing students' humanistic quality. Methods Using dynamic evaluation theory, from three aspects of the evaluational main body, evaluation content and evaluation way, we assessed 261 nursing students' humanistic quality of the five-year undergraduate. Results Based on students' four consecutive years of assessment,results showed that the students' spirit had obviously changed, the students' comprehensive achievements had obviously promoted, the students' employment was obviously higher than other professionals and the students' degree of satisfaction from employing unit had increased yearly. Conclusions Dynamic evaluation can evaluate the best development of students, to strengthen nursing students' humanities education, to improve level of nursing students' humanities education, it is feasible in humanities education of higher nursing profession.