AIM To investigate whether there is vasodilator nerve innervation in rat hind limb and what the nature of vasodilator nerve is. METHODS Wistar rats were treated with reserpine 1 mg?kg -1 ip at 24 h before experiment. The rats were pithed and the hind limb vascular bed was perfused with Krebs-Henseleite solution containing 1 mmol?L -1 phenylephrine at 2 ml?min -1 speed. The hind limb perfused pressure (HPP) as a main index was continuously recorded. Spinal cord electrical stimulation (SES) was repeatedly applied via an electrode at L 1～2 level of lumber vertebra. The various tool drugs were administered by iv or infusion by added to persusion solution. The data is expressed as decrease percentages of HPP increased by continuous infusion of phenylephrine. RESULTS HPP was increased from (5 7?1 5) to (21 6?3 7) kPa ( n =37) after phenylephrine perfusion. SES caused a fall of HPP in frequency dependent and voltage dependent manner. An optimum parameters of SES (10 Hz, 50 V and 1 msec) was selected to observe effects of various tool drugs on depressor response of HPP to SES. Tetrotodoxin (0 3 ?mol?L -1 ) abolished the effect completely. L NAME (10 ?mol?L -1 ), a NO synthase inhibitor, had no effect. Ganglion blocker arfonad (110 mg?kg -1 , iv, M R blocker atropine (10 ?mol?L -1 ), ? receptor blocker propranolol (1 ?mol?L -1 ) and P 1 receptor blocker aminophylline (10 ?mol?L -1 ) had also no effect. Glibenclamide (0 1 mmol?L -1 ), an ATP sensitive K + channel blocker, markedly abolished and slightly reversed the effect. CONCLUTION The nonadrenergic noncholinergic vasodilator nerve exists in rat hind limb and is not nitroxidergic or purinergic nerve. This nerve may be peptidergic nerve which releases some peptide such as CGRP and the major mechanism of vasodilatation probably activates the ATP sensitive K + channel.

Objective To investigate the protective effect of nimodipine on neuron of the rats with focal cerebral ischemia-reperfusion injury and the expressions of Bax and Bcl-2,and to clarify their mechanisms.Methods The focal cerebral-ischemia reperfusion model was induced by the middle cerebral artery occlusion(MCAO)method. 30 male Wistar rats were randomly divided into sham operation,model,and nimodipine groups(n=10).The neurological deficit score was performed after 2 h ischemia following 2 h reperfusion.The infarction was observed by TUNEL staining and the expressions of Bax and Bcl-2 were detected by SP immunohistochemistry method. Results Compared with model group, the number of apoptotic cells of the rats in nimodipine group was significantly decreased(P<0.05),the expression of Bax was significantly decreased (P<0.05),and the Bcl-2 expression was increased significantly(P<0.05).The morphological examination showed that the neurons of the rats in model group had serious necrosis and edema while the number of dead cells in nimodipine treatment group was reduced and the edema was improved.Conclusion Nimodipine has a protective effect on brain tissue of the rats with focal cerebral ischemia-reperfusion inj ury, which is closely related to the down-regulation of Bax and up-regulation of Bcl-2 and inhibition of the apoptosis of neuron.

Objective To study the effects of different transport protein on the transport of 7,4'-dihydroxyflavone (7,4'-DHF) and its metabolite (7,4'-DHF-S) in Caco-2 cell model.Methods Ultra performance liquid chromatography was employed to determinethe content of 7,4'-DHF and 7,4'-DHF-S incubation buffer,their structures were identified by LC-MS/MS.Bidirectional transport of Caco-2 cells model was used to investigate the influence of ko143 (the inhibitor of BCRP) and MK571 (the inhibitor of MRP2) on the transport of 7,4'-DHF and 7,4'-DHF-S,respectively.Results Metabolic product of 7,4'-DHF in Caco-2 monolayer cell was identified as one monosulfate;PDR of 7,4'-DHF was (1.43 ± 0.11),PDR of ko143 and MK571 on the apparent permeability of 7,4'-DHF was (1.59 ± 0.04) and (1.48 ± 0.07) (P ＞ 0.05);PDR of 7,4'-DHF-S was (1.60 ± 0.06);ko143 could significantly reduce the apparent permeability of 7,4'-DHF-S,and the PDR was (0.23 ±0.03) (P ＜ 0.01);MK571 had no significant effect on the apparent permeability of the 7,4'-DHF-S,and the PDR was (1.51±0.04) (P ＞ 0.05).Conclusion Caco-2 cells can mediate the suffonated reaction of 7,4'-DHF;7,4'-dihydroxyflavone sulfonated combination product may be a substrate for BCRP.

Purpose To compare pharmacokineics of puerarin and crude extract in rats.Methods Rats received 500 mg/kg puerarin and puerarin crude extract by oral administration respectively.Hydroxybenzoic acid was selected as internal standard and the plasma concentration of the puerarin and crude extract was analyzed by HPLC.The pharmacokinetics parameters were calculated with DAS2.0.Results The pharmacokinetics of puerarin and puerarin crude extract was both best fitted with two-compartment models in rats after oral administration,and the pharmacokinetics main parameters of the two formulations were different:the AUC_(0-t) and C_(max) of puerarin were much greater than those of puerarin crude extract,but T_(max),t_(1/(2z)),CL/F and V_z/F were much lesser than those of puerarin crude extract.Conclusion The complex components in pueraria crude extract can affect the pharmacokinetics of puerarin in rat in vivo.

Objective To investigate the kinectics characteristics of sulfation of apigenin mediated by SULTIA3.Methods After incubation of apigenin using in vitro SULT1A3 system, high-performance liquid chromatography was utilized to determine the sulfates of apigenin.Mass spectrum(MS) were employed to elucidate the structure of metabolite.The program GraphPad Prism 5 was used to perform the kinetic characterization of SULT1A3 catalyzed metabolism of apigenin.Results A liner calibration curve for the assay of apigenin was validated in the range of 0.15625 ~30 μM with the recoveries of at least 80% and intra-day and inter-day RSD of less than 15%.Metabolic product of apigenin and SULT1A3 in the incubated system was identified one monosulfate.The metabolic behavior of apigenin in SULT1A3 was followed substrate inhibition kinetics.Apparent kinetic parameters of metabolism of apigenin by SULT1A3, Kmwas(0.355 ±1.04) μM and Ksi was(23.62 ±0.06) μM,Vmax was(65.71 ±1.30) nmol/(min? mg),Vmax/Km was 185.10 mL/(min? mg).Conclusion SULT1A3 can mediate the binding of apigenin sulfonated reaction, and the character of enzymatic kinetics shows substrate inhibition.Sulfation of apigenin mediated by SULTIA3 may play an important role in phaseⅡmetabolic in vivo.

Objective To investigate the metabolic profile of chrysin in SULT1A3 and human small intestine S9.Methods After incubation of chrysin using in vitro SULT1A3 and human small intestine S9 system, high-performance liquid chromatography was utilized to determine the sulfates of chrysin.Mass spectrum(MS) were employed to elucidate the structures of metabolite.Results In the SULT1A3 with PAPS, Km were (3.06 ±1.04) and (0.41±0.06) μM, Vmax were (12.13 ±1.30) and (6.72 ±1.61) nmol/(min· mg), Vmax/Km were 3.96 and 16.39 mL/(min· mg), respectively.In the human small intestine S9 with PAPS, Km were (1.92 ±0.35) and (0.01 ±0.00) μM, Vmax were (0.52 ±0.02) and (0.08 ± 0.02) nmol/(min· mg), Vmax/Km were 0.27 and 8.00 mL/(min· mg).The metabolic behavior of chrysin in SULT1A3 and human small intestine S9 both were followed biphasic kinetics.The sulfation of chrysin in SULT1A3 showed a significant correlation with that in human small intestine S9(R2 =0.985).Conclusion The result indicates that SULT1A3 is the major enzyme to the metabolism of chrysin, human small intestine may be the main metabolic organs of chrysin.

Objective To investigate the correlation of fibulin 5(FBLN5)and Rho GTPase activating protein 4(ARHGAP4)in gastric cancer tissues and their relationship with the prognosis of patients.Methods One hundred gastric cancer patients admitted to the Department of General Surgery of Shijiazhuang First Hospital were selected for the study,and gastric cancer tissues＞1 cm from the tumor margin and paracancerous tissues 5 cm from the tumor margin were collected.Retrospective analysis of the relationship between FBLN5 and ARHGAP4 expression profiles and clinicopathological indices of gastric cancer,as well as their effects on survival,was performed with univariate and Cox regression analyses of the patients'clinical data.Immunohistochemical staining was used to detect the expression of FBLN5 and ARHGAP4.The relationship between the expression levels of different FBLN5 and ARHGAP4 and the survival time of patients.Results The positive expression rate of ARHGAP4 was lower than that of paracancerent tissues,and the positive expression rate of FBLN5 was higher than that of paracancerent tissues(P＜0.05).ARHGAP4 expression was associated with tumor site,Lauren's staging,lymph node metastasis,TNM stage,differentiation degree,CE,CA19-9,CA125,and immune score(P＜0.05);FBLN5 expression was associated with Lauren's staging,lymph node metastasis,TNM stage,differentiation degree,CE,CA19-9,CA125,and immune score(P＜0.05);Kaplan-Meier analysis showed that the 3-year cumulative survival rate of FBLN5 positive group was,which was lower than that of negative group(P=0.044).The 3-year cumulative survival rate of ARHGAP4 positive patients was higher than that of negative patients(P=0.021).Cox results showed that positive ARHGAP4 was protective factor for survival(P＜0.05).Conclusion The prognosis of patients in the FBLN5-positive group is worse than that in the negative group,high expression of FBLN5 is a risk factor for survival,and the prognosis of patients with ARHGAP4 negative group is better than that in the positive group,which are closely related to the development and prognosis of gastric cancer patients.

Objective:To develop and validate a liquid chromatography-tandem mass spectrometry method for determining levofloxacin in plasma sample from pediatric patients.Method:This is a prospective, observational study. The clinical residual plasma samples from healthy individuals for physical examination in Children's Hospital Affiliated to Zhengzhou University were collected as blank matrix. Plasma samples from five pediatric patients who did not receive levofloxacin or ciprofloxacin in the department of Respiration were collected for methodological evaluation. In addition, 34 clinical plasma samples from 22 pediatric patients (9 males and 13 females; mean age (8.1±3.7) years) using levofloxacin was collected, and their plasma concentrations were determined. Using ciprofloxacin as the internal standard, levofloxacin in plasma samples was quantified by liquid chromatography-tandem mass spectrometry following protein precipitation using acetonitrile. A C18 column (Shim-pac GIST-HP C18, 2.1 mm×100 mm, 3 μm) and mobile phase composed of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) with gradient elution at a flow rate of 0.4 ml/min were used to separate levofloxacin. The column temperature was 40 ℃, injection volume was 1 μl and the total analysis time was 9 min. Levofloxacin and ciprofloxacin were ionized with an ESI source in positive ion mode and detected in multiple reaction monitoring (MRM) mode. The detected ions of levofloxacin and ciprofloxacin were m/z 362.10→318.1 and 332.15→231.05, respectively. The method′s specificity, sensitivity, linearity, precision, accuracy, recovery rate, stability, matrix effect, and carry-over were validated. All statistical analyses were performed with SPSS statistical software (version 17.0). The normality of the data was detected by the K-S test. A P<0.05 was considered statistically significant for two tailed tests. Results:The LC-MS/MS method showed a good linearity within the range of 0.062 5-20 mg/L, with the lower detection limit of levofloxacin of 0.062 5 mg/L. The calibration curve for levofloxacin was Y=0.093X+0.010 ( R2>0.99). Under different quality control levels, the accuracy ranged from 92.57% to 104.39%, and the intra-day and inter-day imprecision ranged from 2.32% to 9.35%. These values were not affected by the normal matrix, 5% hemolysis matrix and 15% hyperlipidemia matrix. Furthermore, the levofloxacin plasma samples were stable in the short term. A total of 34 plasma samples from 22 patients were collected and analyzed. Only 2 plasma samples were below the lower limit of quantification, while the other plasma concentrations of levofloxacin were ranged from 0.091 to 6.755 mg/L. Cmax was (5.52 ± 1.09) mg/L. Conclusion:The LC-MS/MS method meets the requirements of the reference method and requires a small sample size (50 μl), making it suitable for the determination of levofloxacin in plasma from pediatric patients.

Objective:To investigate the effects of gambogenic acid(GNA)on the proliferation and apoptosis of CAL27 cell xenograft tumor in nude mice.Methods:18 SPF nude mice were randomly divided into 3 groups(n=6).All nude mice were inoculated with CAL27 cells at logarithmic growth stage to establish subcutaneous transplanted tumor models.The mice in low and high dose GNA groups were treated with GNA of 8.0 mg/kg iv.and 16.0 mg/kg iv.every other day,respectively,and those in the control group was given the same amount of normal saline.The tumor growth curve was plotted during drug administration.2 weeks later,the nude mice were sacrificed,the tumor tissue was removed and the tumor inhibition rate was evaluated by the tumor size measurements.TUNEL as-say was used to detect the apoptosis of transplanted tumor cells in the groups.The expression levels of AKT,Bcl-2 and PI3K proteins in tumor tissue were detected by immunohistochemistry(IHC).The toxicity and side effects of GNA on normal tissues were detected by HE staining.Results:The transplanted tumors in low and high dose GNA groups grew slowly,and the tumor weight and volume were significantly lower than those in the control group(P＜0.05),the tumor inhibition ratio of low and high dose groups was 57.58％and 83.68％respectively.TUNEL results showed that the apoptosis index of GNA low and high dose groups was higher that of control group(P＜0.05).IHC results showed that the expression of AKT,Bcl-2 and PI3K in the tumor tissues of nude mice in low and high dose GNA groups was lower than that in the control group(P＜0.05).HE results showed that GNA had not effect on normal tissues and or-gans(P＜0.05);Conclusion:GNA may induce CAL27 cell apoptosis by regulating the expression of AKT,Bcl-2 and PI3K,and in-hibites the development of human tongue squamous cell carcinoma with little effect on normal tissues and organs.

Objective: To investigate the effect of siRNA silencing α1 antitrypsin (α1-AT) gene on the biological behavior of rheumatoid arthritis fibroblast-like synoviocytes (RA-SFs). Methods: Primary culture of knee synovial tissue from 5 patients with rheumatoid arthritis (RA) was performed. The artificially synthesized silencing α1-AT siRNA specifically inhibits the expression of α1-AT in RA-SFs. After 24 and 36 hours of transient transfection, the inhibition efficiency was detected by Quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the expression of related genes after α1-AT gene silencing was detected.Furthermore, ethyl thiazolyl tetrazolium (MTT) assay, Trans-well chamber, cell scratch and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of interfering α1-AT expression on cell proliferation, invasion and migration, and secretion of interleukin (IL)-17, Tumor necrosis factor (TNF)-α, IL-1α, IL-1β and other related inflammatory factors. At the same time, when the pathway inhibitor (ERK inhibitor, signal transducer and activator of transcription 3 (STAT3) inhibitor, NF-κB inhibitor) stimulated cells, the effect on α1-AT was changed. One-way analysis of variance was used for comparison between the two groups; further pairwise comparison using LSD-t test; The count data was checked by χ² test. Results: Compared with the negative control group, the siRNA α1-AT group was transfected for 24 hours, the α1-AT mRNA level was significantly inhibited (P<0.05), and the proliferation rate of RA-SFs was not affected (P>0.05), and the invasion and migration ability of cells decreased significantly (P<0.05). The secretion of IL-17 and IL-1β was slightly decreased (P>0.05), while TNF-α and IL-1α were not affected (P>0.05). In addition, the expression of α1-AT downstream genes Matrix metallop roteinases (MMP)-2 169, Hu-Bax1, MMP-9, Hu Bax and Hu Bcl-xl were significantly decreased (P<0.05). Moreover, the signaling pathway inhibitors ERK inhibitor (PD98059) and NF-κB inhibitor (PDTC) had significant effects on α1-AT (P<0.05). Conclusion The α1-AT gene silencing has potential effect on the biological behavior of RA SFs. Further study of this mechanism is helpful to provide evidence for the treatment of RA.