The study aims to develop an LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in urine samples, and to investigate their urinary excretion characteristics in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection (HTLPI). The urine samples were extracted by methanol, and then separated on a Hedera ODS-2 column with a mobile phase of acetonitrile and 5 mmol · L(-1) ammonium acetate buffer solution containing 0.05% formic acid (20:80). Electrospray ionization source was applied and operated in the positive ion mode using MRM. The method exhibited good linearity over the concentration range of 0.03 -40 µg · mL(-1). The values on both the occasions (intra- and inter-day) were all within 15% at three concentration levels. No matrix effect and carry-over effect were observed. Amygdalin and paeoniflorin were stable in human urine under different storage conditions. Approximately 79.6% of the administered amount of amygdalin was excreted unchanged in urine within 24 h and which was 48.4% for paeoniflorin. The developed LC-MS/MS method can be applied to evaluate the urinary excretion of amygdalin and paeoniflorin.

Objective To study the effect of transcutaneous electrical acupoint stimulation(TEAS) on endtidal concentration of sevoflurane in upper abdominal operation.Methods The use of prospective,randomized,blinded principles.A total of 50 patients underwent selective epigastric operations with ASA Ⅰ - Ⅱ were randomly divided into group A(25 cases) and B(25 cases).In group A,TEAS was performed and sevoflurane was inhaled during operation.In group B,only sevoflurane was inhaled and TEAS was not performed during operation.Electrical stimulation on Nei-guan,He-gu and Zu-sanli was performed for 30min before induction of anesthesia in group A and meanwhile patients in group B were waiting for 30min in operating room.After intubation,in group A,TEAS was performed persistently and sevoflurane was inhaled,meanwhile remifentanil was infused persistently during operation.In group B,only sevoflurane was inhaled and remifentanil was infused persistently during operation.At time points:before TEAS( T0 ),skin incision( T1 ),exploratory laparotomy( T2 ),30min after exploratory laparotomy ( T3 ),60min after exploratory laparotomy( T4 ),blood glucose and angiotensin Ⅱ were measured,recorded 10min after the start of surgery,once for each end-tidal sevoflurane concentration.Results End-tidal concentration of sevoflurane of group A ( 1.4 ± 0.2 ) ％,was significantly lower than group B( 1.9 ± 0.4 ) ％ ( t =3.147,P ＜ 0.01 ).Cortisol and angiotensin Ⅱ were increased at T1 and T2 compared with T0 in both groups(F =2.256,2.432,2.132,2.334,all P＜0.05).Cortisol and angiotensin Ⅱwere decreased in group A compared with in group B at T1 ～T4(t =2.159,2.232,2.453,2.602,al1 P ＜0.05).Conclusion TEAS can decrease end-tidal concentration of sevoflurane and stress response.TEAS combined with sevoflurane has synergistic effect on general anesthesia.

Objective To evaluate the effect of mild hypothermia on the activity of hippocampal pro tein kinase R-like endoplasmic reticulum kinase (PERK) in a mouse model of cerebral ischemia-reperfusion (I/R).Methods One hundred and twenty male C56BL6 mice,weighing 20-30 g,aged 7 weeks,were randomly divided into 3 groups (n=40 each) using a random number table:sham operation group (group S),I/R group,and mild hypothermia group (group H).Cerebral I/R was induced by occlusion of bilateral common carotid arteries for 15 min followed by reperfusion in anesthetized mice.In group H,surface cooling was performed immediately after reperfusion,and the rectal temperature was maintained at 32-34 ℃ for 3 h.In I/R and S groups,the rectal temperature was maintained at 36.8-37.2 ℃.At 6,12,24 and 72 h of reperfusion,10 mice were sacrificed in each group,and the hippocampi were removed for determination of the number of apoptotic neurons in hippocampal CA1 region (by TUNEL),and phosphorylated PERK (p-PERK) expression (by Western blot).Results Compared with group S,the number of apoptotic neurons was significantly increased,and the expression of p-PERK was up-regulated at each time point in I/R and H groups (P＜0.05).Compared with group I/R,the number of apoptotic neurons was significantly decreased,and the expression of p-PERK was downregulated at each time point in group H (P＜0.05).Conclusion Mild hypothermia can reduce endoplasmic reticulum stress through inhibiting hippocampal PERK activity,thus attenuating cerebral injury in a mouse model of cerebral I/R.

Objective To evaluate the effect of PUN282987 on global cerebral ischemia-reperfusion (I/R) injury in aged rats.Methods One hundred and twenty pathogen-free healthy male SpragueDawley rats,aged 18-22 months,weighing 450-600 g,were divided into 3 groups (n=40 each) using a random number table:sham operation group (group S),global cerebral I/R group (group I/R) and α7 nicotinic acetylcholine receptor (α7nAChR) agonist PNU282987 group (group PUN).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 0.4 ml/100g,and global cerebral I/R was produced by 4-vessel occlusion technique in I/R and PUN groups.PUN282987 2.4 mg/kg was injected intraperitoneally before ischemia in group PUN.At 1,5,12 and 24 h of reperfusion,10 rats were randomly selected in each group and then sacrificed,and the brains were removed for detection of the neuronal apoptosis and for determination of the expression of α7nAChR,choline acetyltransferase (ChAT),tumor necrosis factor-α (TNF-α) and intedeukin-1β (IL-1β) in the hippocampal CA1 region.Apoptosis rate was calculated.Results Compared with group S,the apoptosis rate was significantly increased,and the expression of α7nAChR,ChAT,TNF-α and IL-1β was up-regulated at each time point in I/R and PUN groups (P＜0.05).Compared with group I/R,the apoptosis rate was significantly decreased,the expression of α7nAChR and ChAT was up-regulated,and the expression of TNF-α and IL-1β was down-regulated at each time point in group PUN (P＜0.05).Conclusion PUN282987 can reduce global cerebral I/R injury in aged rats.

Objective To evaluate the effect of parecoxib on the inflammatory response after radical lung cancer surgery in elderly patients.Methods Forty-one ASA Ⅱ or Ⅲ elderly patients of both sexes,aged 65-78yr,weighing 52-81 kg,undergoing elective radical lung cancer surgery performed via a thoracoscope,were randomly divided into control group (group C,n =20) and parecoxib group (group P,n =21).Tracheal intubation was performed after induction of anesthesia.Anesthesia was maintained with intravenous infusion of remifentanil and propofol,inhalation of sevoflurane and intermittent intravenous boluses of vecuronium.Bispectral index value was maintained at 40-50.Group P received intramuscular parecoxib sodium 40 mg at 30 min before anesthesia and at 12,24 and 36 h after surgery.The equal volume of normal saline was given in group C.Both groups received patient-controlled intravenous analgesia with morphine after operation.VAS scores at rest were maintained ≤ 3.The consumption of morphine and Ramsay sedation score at 12,24 and 48 h after operation were recorded.Blood samples were taken from the central vein before surgery (T0),at the end of surgery (T1) and at 12,24 and 48 h after surgery (T2-4) for determination of plasma TNF-α and IL-8 concentrations.Blood samples were collected from the radial artery at T0-4 for determination of PaO2.Alveolar-arterial oxygen tension difference and oxygenation index were calculated.Pulmonary complications were recorded within 2 days after surgery.Results The consumption of morphine,Ramsay sedation score,plasma TNF-α and IL-8 concentrations,alveolar-arterial oxygen tension difference and incidence of pulmonary complications were significantly lower,and oxygenation index was significantly higher in group P than in group C (P ＜ 0.05).Conclusion Parecoxib can reduce the inflammatory response after radical lung cancer surgery in elderly patients,thus improving respiratory function and reducing pulmonary complications after surgery.

Objective To evaluate the changes in cholinergic anti-inflammatory pathway in hippocampi global in aged rats with cerebral ischemia/reperfusion (I/R ) injury .Methods One hundred and twenty male Sprague-Dawley rats , aged 18-22 months ,weighing 450-600 g ,were randomly divided into 2 groups ( n= 60 each):sham operation group (group S) and global cerebral I/R group (group I/R) .The animals were anesthetized with intraperitoneal 10% chloral hydrate 0.4 ml/100 g .Global cerebral I/R was induced by 4-vessel occlusion method described by Pulsinelli .Fifteen rats were sacrificed at 1 ,3 ,5 and 7 days of reperfusion ,and brains were removed for determination of neuronal apoptosis and expression of α7 nicotinic acetylcholine receptor (α7nAChR ) , choline acetyltransferase (ChAT ) ,tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in the hippocampal CA1 region .The apoptosis rate was calculated .Results Compared with group S ,the apoptosis rate was increased and the expression of α7nAChR ,ChAT ,TNF-αand IL-1βwas up-regulated in group I/R ( P<0.05 or 0.01 ) . The expression of α7nAChR and ChAT was up-regulated gradually during reperfusion and peaked at 5 day of reperfusion ( P< 0.05 ) .Conclusion Global cerebral I/R injury can activate cholinergic anti-inflammatory pathway in aged rat hippocampi ,and the activation of this pathway is the endogenous mechanism of inhibition of excessive inflammatory responses in brain tissues .

Objective To investigate the efficacy and pregnancy outcomes of women receiving double-catheter epidural block in labor analgesia, and compare the results with single-catheter epidural block.Methods A double-blind clinical trial was conducted on 206 full-term singleton primiparas, aged 25-35 and at the 37 -42 weeks of gestation who delivered at the Department of Obstetrics, Qingdao Municipal Hospital from August 2006 to December 2008, which were randomly divided into two groups:double-catheter epidural block ( group D, n = 103) and single-catheter epidural-block ( group S, n = 103 ).Women in group D were given mixture of 0.1% repivacaine hydrochloride and 0.5 mg/L sufentinil 4 -6 ml as initial dose.Patient control epidural analgesia pump (PCEA) was connected with the upper catheter after 45 minutes.A bolus dose of 4 -6 ml analgesia mixture was infused according to the condition through the lower catheter.Women in group S received analgesia mixture 10 - 15 ml as initial dose and PCEA pump was connected after 45 minutes.Oxytocin was infused in both groups according to uterine contraction after 30 minutes.The following indexes was observed: ( 1 ) visual analogue scales (VAS); (2) modified Bromage Scores;(3) the total dose of analgesia mixture, the percentage of oxytocin infusion, duration of labor and duration of the second stage of labor; (4) fetal birth weight and Apgar scores( 1,5 minutes); (5) mode of delivery; (6) the concentration of plasma cortisol and angiotension Ⅱ at the beginning of regular uterine contraction and at the time when cervical dilated to 4 cm and 10 cm and fetal disengagement; (7)anesthesia-related complications.Results ( 1 )The neonatal birth weight and Apgar scores ( 1,5 minutes)of group D were (3456 ±468)g, 9.8 ±0.6 and 9.9 ±0.7, respectively, while(3399 ±569) g, 9.8 ±0.5 and 9.9 ±0.7 in group S( P ＞0.05).No motor function block was reported in any group and the modified Bromage score was zero.(2) The total dose of analgesia mixture in group D was similar to that in group S [(57 ±9) ml vs.(58 ±11) ml, P＞0.05].However, the percentage of women received oxytocin in group D was smaller [59.2% (61/103) vs.81.6% (84/103), P ＜ 0.01], and the total time of labor and the duration of second stage of labor in group D were shorter[(532 ± 140) minutes vs.(608 ± 150) minutes;(46 ± 31 ) minutes vs.(60 ± 34) minutes, P ＜ 0.05].(3) There were no significant differences in VAS at 30 minutes after initial dose and in the first stage of labor between group D and S ( 1.2 ± 1.1 vs 1.2 ± 1.1,1.1 ± 1.1 vs.1.2 ± 1.0, P＞0.05).VAS at the second stage of labor stage was lower in group D than in group S ( 1.2 ± 1.1 vs.4.5 ± 2.2, P ＜ 0.01 ).(4) The rate of cesarean section, instrumental delivery and episiotomy in group D were lower than in group S (7.8% vs.17.5%, 7.8% vs.15.5%, 10.7% vs.18.4%, P ＜ 0.05).The incidence of fetal distress and meconium-stained amniotic fluid as the indication of cesarean section were similar between the two groups (P ＞ 0.05 ).Lower incidence of fetal malpresentation and arrested second stage of labor were shown in group D than in group S (2.9% vs.9.7%, 1.0% vs.5.8%, P ＜ 0.05 ).(5) The concentration of plasma cortisol and angiotension Ⅱ were lower in group D than in group S [(86 ±25) ng/L vs.( 100 ±20) ng/L, (278 ±53) nmol/L vs.(311 ±53)nmol/L, P＜0.05] only at the end of second stage of labor, but not at any other times(P ＞0.05).(6) No serious anesthesia-related complications were reported in any groups.Some light backache around the puncture point were complained by 29.1% (30/103) of the women in group D and 31.1% (32/103) in group S(P ＞0.05).Conclusion Double-catheter epidural block can provide better analgesia effect during labor than single-catheter epidural block, without any adverse influence on delivery outcomes.

Objective To evaluate the effect of electroacupuncture (EA) preconditioning on inositol-requiring kinase 1 (IRE1)-X-box binding protein 1 (XBP1) signaling pathway in endoplasmic reticulum in the cortex in a rat model of cerebral ischemia-reperfusion (I/R).Methods One hundred and eight pathogen-free healthy male Sprague-Dawley rats,aged 8-12 weeks,weighing 200-250 g,were assigned into 3 groups (n =36 each) using a random number table:sham operation group (group S),group I/R and EA preconditioning group (group EA).Focal cerebral I/R was induced by occlusion of right middle cerebral arteries for 2 h followed by reperfusion in rats anesthetized with chloral hydrate.In group EA,Baihui acupoints were stimulated with an electric stimulator for 30 min once a day for 5 consecutive days starting from 5 days before ischemia,and the model was established at 24 h after the last preconditioning.Rats were sacrificed after neurological deficit was scored at 6,12 and 24 h of reperfusion,brains were removed,and the ischemic area in cerebral cortex was isolated for examination of the cell ultrastructure (with an electronic microscope) and for determination of the expression of IRE1 and XBP1 (by Western blot).Results Compared with group S,the neurological deficit scores were significantly increased,and the expression of IRE1 and XBP1 in the ischemic area was up-regulated at each time point in I/R and EA groups (P＜0.01).Compared with group I/R,the neurological deficit scores were significantly decreased,and the expression of IRE1 and XBP1 was up-regulated at each time point in group EA (P＜0.05).The cell damage in the ischemic area in cerebral cortex was significantly attenuated in group EA when compared with group I/R.Conclusion The mechanism by which EA preconditioning attenuates cerebral I/R injury is related to activating IRE1-XBP 1 signaling pathway and relieving endoplasmic reticulum stress in rats.

Objective:To evaluate the obesity factor on ventilator-induced lung injury (VILI) in rats.Methods:Forty-five clean-grade male Sprague-Dawley rats, aged 6-8 weeks, were divided into 3 groups ( n = 15 each)according to the body weight: normal weight control group (group C), normal weight VILI group (group CV) and obese VILI group (group FV). The body weight was 233-267 g in C and CV groups and 288-332 g in FV group.In group C, the tidal volume (V T) was 10 ml/kg.In CV and FV groups, the rats were ventilated for 4 h with the V T set at 40 ml/kg, respiratory rate 40 breaths/min, inspiratory/expiratory ratio 1∶2, PEEP 0 mmHg, and fraction of inspired oxygen 21% to establish the VILI model.The arterial blood samples were collected immediately before tracheal intubation and at 4 h of mechanical ventilation for blood gas analysis and PaO 2 recording.The remaining blood samples were used for plasma collection.The rats were sacrificed after blood collection at 4 h of ventilation, and the bilateral lung tissues were isolated to collect the bronchoalveolar lavage fluid (BALF). The concentrations of leptin in plasma and tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interleukin-6 (IL-6) in plasma and BALF were detected by enzyme-linked immunosorbent assay.The wet/dry weight (W/D) ratio of lung tissues was measured.The pathological changes of lung tissues were observed after HE staining, and the lung injury score was evaluated.The expression of NF-κB p65 in lung tissues was detected by Western blot. Results:Compared with group C and group CV, the plasma leptin concentration was significantly increased in group FV( P<0.01). Compared with group C, the concentrations of TNF-α, IL-6 and IL-1β in plasma and BALF were significantly increased, PaO 2 was decreased, the lung injury score and W/D ratio of lung tissues were increased, and NF-κB p65 expression was up-regulated at 4 h of ventilation in CV and FV groups ( P<0.01). Compared with group CV, the concentrations of TNF-α, IL-6 and IL-1β in plasma and BALF were significantly decreased, PaO 2 was increased, the lung injury score and W/D ratio of lung tissues were decreased, and NF-κB p65 expression was down-regulated at 4 h of ventilation in group FV ( P<0.05). Conclusion:Obesity factor can reduce VILI in rats, and the mechanism may be related to the increase in plasma leptin levels.

Objective To evaluate the effect of penehyclidine hydrochloride on the expression of airway mucin 5AC(MUC5AC)during ventilator-induced lung injury(VILI)and the relationship with Toll-like receptor 4/myeloid differentiation factor 88(TLR4/MyD88)signaling pathway in rats.Methods Thir-ty-six clean-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 200-250 g,were divided into 3 groups(n＝12 each)using a random number table method: sham operation group(group S),VILI group(group VILI),and penehyclidine hydrochloride group(group P).The rats were tracheotomized in group S.The rats were tracheotomized,connected to a small animal ventilator and mechanically ventilated for 4 h with the tidal volume of 20 ml/kg,respiratory rate 80 breaths/min,inspiratory/expiratory ratio 1 ∶1,and inspired oxygen fraction ratio 21％in VILI and P groups.At 30 min before mechanical ventilation,penehyclidine hydrochloride 2 mg/kg was injected via the tail vein in group P,and the equal volume of nor-mal saline was given instead in S and VILI groups.At 4 h of mechanical ventilation,the arterial blood sam-ples were taken for measurement of PaO2.The rats were then sacrificed,and broncho-alveolar lavage fluid(BALF)was collected for determination of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)concentrations by enzyme-linked immunosorbent assay.The lung specimens were collected for calculation of the wet/dry weight ratio(W/D ratio),for examination of pathological changes which were scored after haematoxylin and eosin staining(under a light microscope),and for determination of the ex-pression of MUC5AC(by immunohistochemistry),expression of TLR4,MyD88,p38 mitogen-activated protein kinase(p38MAPK)and nuclear factor-kappa B(NF-κB)in lung tissues(by Western blot),and expression of MUC5AC mRNA in lung tissues(by real-time polymerase chain reaction).Results Com-pared with group S,PaO2 was significantly decreased,the W/D ratio and lung injury score were increased,the expression of MUC5AC protein and mRNA was up-regulated,the concentrations of IL-1β,IL-6 and TNF-α in BALF were increased,and the expression of TLR4,p38MAPK,MyD88 and NF-κB was up-reg-ulated in VILI and P groups(P＜0.01).Compared with group VILI,PaO2 was significantly increased,the W/D ratio and lung injury score were decreased,the expression of MUC5AC protein and mRNA was down-regulated,the concentrations of IL-1β,IL-6 and TNF-α in BALF were decreased,and the expression of TLR4,p38MAPK,MyD88 and NF-κB was down-regulated in group P(P＜0.05).Conclusion Penehy-clidine hydrochloride can decrease the expression of airway MUC5AC during VILI,and the mechanism may be related to inhibiting activation of TLR4/MyD88 signaling pathway in rats.