Objective To investigate the effects of ketamine on endotexin-induced production of proinflammatory cytokines(IL-6, TNF-?) and activation of their medulating factor NF-?B in vivo. Methods Forty adult male SD rats weighing 200-250 g were randomly divided into 4 groups: (Ⅰ)control group(n=10); (Ⅱ) endotoxin group received intravenous endotoxin(Escherichia coli O111: B4, Sigma) 5 mg?kg~(-1)(n=10); (Ⅲ, Ⅳ)endotexin+ketamine group received ketamine or 5 or 10mg?kg~(-1)?h~(-1) after endotoxin(n=10). The animals were anesthetized with urethane i.p. (1g?kg~(-1)). Carotid artery was cannulated for BP and HR monitoring and jugular vein was cannulated for fluid or drug administration. Two hours after endotoxin administration the animals were sacrificed by exsanguination. Blood was collected and peripheral blood monocytes(PBMC) were isolated. NF-?B activity in PBMC was measured by EMSA and plasma TNF-? and IL-6 levels were determined by ELISA. Results Progressive hypotension and tachycardia developed after endotoxin administration. Endotexin also increased NF-?B activity in PBMCs and plasma TNF-? and IL-6. Ketamine 10 mg?kg~(-1) attenuated the endotexin-induced hemedynamie levels. Ketamine(5, 50 mg?kg~(-1)?h~(-1)) suppressed NF-?B activity in PBMC and inhihited plasma TNF-? level but plasma IL-6 level was not affected. Conclusion Ketamine can suppress endotoxin-induced NF-?appa B activation. Subanesthetic dose of ketamine has anti-inflammatory action.

Objective To evaluate the regulatory role of acetylcholine receptor in muramyl dipeptide (MDP)-induced activation of Nod-like receptor 2/receptor-interacting protein 2 (2NLR2/RIP2) pathway in macrophages of mice.Methods RAW264.7 cells at the logarithmic growth phase were seeded in 12-well plates (density 1 × 106 cells/ml,2 ml/well),a total of 108 wells.The cells were randomly divided into 3 groups (n =36 each) using a random number table:control group (group C),MDP group (group M),and GTS-21 (a7nAChR specific agonist) group (group G).The cells were routinely cultured in group C.MDP with the final concentration of 10 μg/ml was added to the culture medium in group M.MDP with the final concentration of 10μg/ml and GTS21 with the final concentration of 50 μg/ml were added to the culture medium in group G.The cells were incubated for 24 h.At 1,6 and 24 h of incubation with MDP,12 wells were chosen and the cell suspension was obtained for measurement of NLR2 mRNA expression (by real-time fluorescent quantitative PCR),RIP2 expression (by Western blot),and concentrations of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) in the culture media (by ELISA).Results Compared with group C,the levels of NLR2 mRNA,RIP2,TNFα and HMGB1 were significantly increased at each time point in group M (P ＜ 0.05).Compared with group M,the levels of NLR2 mRNA,RIP2,TNF-α and HMGB1 were significantly decreased at each time point in group G (P ＜ 0.05).Conclusion Acetylcholine receptor can suppress MDP-induced transduction of NLR2/RIP2 pathway in macrophages of mice.

Objective To investigate the effect of electro-acupunctare at zusanli on acute lung injury in a rat model of sepsis after scald.Methods Fifty SPF male Sprague-Dawley rats,weighing 200-250 g,aged 2-3 months,were randomly divided into 5 groups (n =10 each) using a random number table:control group (group C),sepsis after scald group (group SS),electro-acupuncture at zusanli group (group E),electric stimulation of non-acupoint group (group NE) and electro-acupuncture at zusanli + α-bungarotoxin (α-BGT,a selective α7 nicotinic acetylcholine receptor antagonist) group (group α-BGT).The rats were subjected to a third degree scald covering 20％ total body surface (TBS) and muramyl dipeptide (MDP) 5 mg/kg was injected into the femoral vein immediately after scald to induce sepsis.Electro-stimulation (3 V,2 ms,3 Hz) of bilateral zusanli was performed for 12 min starting from the time point immediately after MDP injection and every 8 h for 2 consecutive days in group E.In group NE,electro-stimulation was performed at the points 5 mm lateral to the bilateral acupoints of Zusanli and the method was similar to those previously described in group E.In group α-BGT,α-BGT 1.0 μg/kg (in 1 ml of normal saline) was injected into the femoral vein before electro-stimulation of zusanli.At 48 h after treatment,arterial blood samples were obtained for determination of serum tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) protein levels (by ELISA) and lung specimens were removed for microscopic examination and for determination of the expression of Nod like receptor 2 (NLR2) mRNA (by RT-PCR) and receptor interacting protein 2 (RIP2) in the lung tissues (by Western blot).Results Compared with group C,the expression of NLR2 mRNA and RIP2 was significantly up-regulated,and the serum TNF-α and HMGB1 levels were increased in SS,NE and α-BGT groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in group E (P ＞ 0.05).Compared with group SS,the expression of NLR2 mRNA and RIP2 was down-regulated,and the serum TNF-α and HMGB1 levels were decreased in group E (P ＜ 0.05),and no significant change was found in the parameters mentioned above in NE and α-BGT groups (P ＞ 0.05).Compared with group E,the expression of NLR2 mRNA and RIP2 was significantly up-regulated,and the serum TNF-α and HMGB1 levels were increased in NE and α-BGT groups (P ＜ 0.05).The pathological changes of lung tissues were significantly reduced in group E as compared with group SS.Conclusion Electro-acupunctare at Zusanli can reduce acute lung injury in a rat model of sepsis after scald and inhibition of NLR2/RIP2 signaling pathway and activation of cholinergic anti-inflammatory pathway in lung tissues may be involved in the mechanism.

The primary structure of rhFK506-Binding Protein (FKBP) 12 was studied with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) by measuring its molecular weight and tryptic peptide mass fingerprinting.The calculated molecular weight of rhFKBP12 was 11819.5 and the measured value was 11818.0. The relative error was only 0.01%. The measured peptide mass fingrprinting of rhFKBP12was identical with that deduced from its amino acid sequence. It proved that the primary structure of rhFKBP12 was correct and there was no amino acid deletion, mutation and modification in its expression, refolding andpurification.

Objective To identify the correlation factors associated with cerebral intragenic ischemia after temporary parent arterial occlusion in intracranial aneurysm surgery. Methods One hundred and eighteen patients who underwent temporary arterial occlusion in the 120 aneurysms (from a group of 324 consecutive aneurysm patients treated from 1996 to 2002) were reviewed retrospectively. These variables included sex, age, presence of preoperative subarachnoid hemorrhage (SAH), neurological clinical grade, operational timing, duration of arterial occlusion, numbers of temporary occlusion, mode of arterial occlusion, intraoperative aneurysm rupture, hypertension, the location of temporary occlusion, aneurysm size, hyperglycemia, atheromatous mass. Univariate and multivariate were used to investigate the relationship between the variates and postoperative ischemic changes. Results The total times of temporary occlusion were 156, with an average of 1.30. The duration of arterial occlusion ranged from 1 to 45 min (9.75?7.75). Seventeen patients (14.4%) demonstrated evidence of new infarction in the vascular territory subjected to temporary arterial occlusion. Conclusion In the univariate analysis, age, presence of preoperative SAH, duration of arterial occlusion, atheromatous mass are all significantly correlated with postoperative ischemic injuries. Multivariate logistic regression revealed that the age, older more than 60 (P= 0.010 3 , relative risk=4.335), and the duration of arterial occlusion, lasting more than 20 min (P= 0.032 9 , relative risk=4.177), have significant correlation with the injuries. Based on these findings, temporary occlusion is safe and useful in aneurysm surgery and the postoperative cerebral ischemia is less likely to occur when the duration of clipping is shorter than 20 min.

Objective To investigate the effect of nicotine on coagulation abnormalities in endotoxemic rats.Methods Ninety-six male SD rats weighing 200-250 g were randomly divided into 4 groups (n=24 each): group normal saline (group NS);group LPS;group nicotine(group NIC)and group α-bungarotoxin (α7 nicotinic acetylcholine receptor antagonist, group α-BGT) . Endotoxemia was induced by LPS 10 mg/kg injected via femoral vein in LPS, NIC and α-BGT groups. In group NIC nicotine 400 μg/kg was injected intraperitoneally at 30 min before LPS injection. In group α-BGT α-BGT 1 μg/kg was injected intraperitoneally at 15 min before intraperitoneal nicotine. Prothrombin time(PT),activated partial thromboplastin time(APTT),fibrinogen(Fib),antithrombin (AT),von Willebrand factor(vWF),plasminogen activator inhibitor-1(PAI-1),D-dimer,platelet count and TNF-α were measured before (baseline) and 2, 4 and 6 h after LPS injection.Results PT and APTT were significantly prolonged and plasma Fib and AT concentrations and platelet count were significantly decreased, while plasma PAI-1, D-dimer, vWF and TNF-α concentrations were significantly increased after LPS administration in group LPS as compared with group NS. Nitotine pretreatment significantly attenuated the LPS-induced changes in group NIC.The effect of nicotine was counteracted by α-BGT. Conclusion Nicotine can attenuate coagulation abnormalities induced by LPS by acting on α7 nicotinic acetylcholine receptor.

Objective To evaluate the effect of penehyclidine hydrochloride on cell apoptosis during acute lung injury (ALI) induced by two-hit in rats.Methods Thirty male SPF Sprague-Dawley rats, aged 8 weeks, weighing 240-270 g, were randomly assigned into 3 groups (n =10 each) using a random number table: sham operation group (group Sham), ALI induced by blunt chest trauma and hemorrhagic shock group (group ALI), and penehyclidine hydrochloric group (group PHC).In ALI and PHC groups, the rats were subjected to the combination of chest trauma and hemorrhage (mean arterial pressure 35-40 mmHg mm Hg, lasting for 60 min) to establish a model of ALI.In group PHC, penehyclidine hydrochloric 2 mg/kg was injected intraperitoneally at 1 h before blunt chest trauma.At 8 h after successful establishment of the model, the rats were sacrificed, and lungs were removed for examination of the pathologic changes and for determination of Bax, Bcl-2 and caspase-3 expression (using Western blot), tumor necrosis factor-alpha (TNF-α) content (by enzyme-linked immunosorbent assay) , and cell apoptosis (by TUNEL).Apoptotic index was calculated.Results Compared with group Sham, the levels of Bax, caspase-3 and TNF-α and apoptotic index were significantly increased, and Bcl-2 expression was down-regulated in ALI and PHC groups.Compared with group ALI, the levels of Bax, caspase-3 and TNF-α and apoptotic index were significantly decreased, and Bcl-2 expression was up-regulated in group PHC.The pathologic changes of lungs were significantly reduced in group PHC than in group ALI.Conclusion Penehyclidine hydrochloride mitigates ALI induced by two-hit through inhibiting cell apoptosis in rats.

Objective:To study the effects of exosomes (EXO) released by adenoid cystic carcinoma SACC-83 cells on the expression of fibroblast activation protein (FAP) in normal human salivary gland stromal fibroblasts (hSGSFs).Methods:ACC exosomes were isolated from SACC-83 cell culture supernatant by using Total Exosome Isolation Reagent.The whole-mount EXO were characterized and assessed by transmission electron microscope and Western Blot.The exosomes were labeled with green fluorescent dye PKH67 and co-cultured with hSGSFs for 48h,followed by staining with Alexa Fluor 594 Phalloidin and DAPI.Mterwards,exsosomes uptake was observed under a laser scanning confocal microscope.After a 48-hour co-culture of SACC-83 exosomes with hSGSFs,the expression of FAP in SACC-83-EXO-treated hSGSFs was investigated by qRT-PCR and Western Blot.Results:The vesicles isolated from SACC-83 cell culture supernatant had the reported size range of 30-100 nm,expressed the exosomal marker CD63 and TSG101.Mter co-culture of hSGSFs with PKH67 labeled SACC-83 exosomes,exosomes were taken up by hSGSFs and FAP expression was elevated in hSGSFs.Conclusion:Exosomes derived from SACC-83 cells can be taken up by hSGSFs and can induce the expression of FAP in hSGSFs.These results suggest that exosomes derived from SACC-83 cells might induce the transformation of normal salivary gland strormal fibroblasts to cancer associated fibroblasts.

Objective To establish a rat model of sepsis induced by muramyl dipeptide (MDP) after scald burn.Methods Fifty SPF male Sprague-Dawley rats,aged 2-3 months,weighing 200-250 g,were randomly divided into 3 groups:control group (group C,n =10),scald group (group S,n =10) and MDP group (n =30).The rats were subjected to a third-degree scald burn covering 20% of total body surface area in groups S and MDP.The rats were only exposed to 20 ℃ water in group C.MDP 5 mg/kg was injected via the femoral vein at 24 h after scald bum in group MDP.Arterial blood samples were collected at 1,6 and 24 h after MDP injection in group MDP,at 24 h after scald burn in group S,or at 24 h after exposure to 20 ℃ water in group C for blood gas analysis and for measurement of white blood cell (WBC) and platelet (Plt) counts,serum aminotransferase (ALT),aspartate transferase (AST),total bilirubin (TB),creatinine (Cr) and blood urea nitrogen (BUN) levels,creatine kinase isoenzyme-MB (CK-MB) activity,and plasma tumor necrosis factor-α (TNF-α),interferon-γ(IFN-γ),interleukin-6 (IL-6),IL-10 and high mobility group box 1 protein (HMGB-1) levels.The rats were sacrificed after collecting blood samples,and heart,liver,lung,and kidney specimens were obtained for microscopic examination of pathologic changes.The activity of myeloperoxidase (MPO) in lung tissues was measured.Another 90 male Sprague-Dawley rats were randomly divided into 3 groups and treated as the method previously described for record of the survival rate within 72 h.Results Compared with C group,the plasma IL-6,IL-10,IFN-γ and HMGB1 levels,WBC count,serum ALT,AST,and BUN levels and MPO activity were significantly increased,and the survival rate within 72 h was decreased in S group,and the plasma TNF-α,IL-6,IL-10,IFN-γand HMGB-1 levels,serum ALT,AST,TB,BUN,Cr and CK-MB levels,MPO activity,PaCO2 and BE value were significantly increased,and WBC and PLT counts,pH value,PaO2 and survival rate within 72 h were decreased in MDP group (P ＜ 0.05).Compared with S group,the plasma TNF-α,IL-6,IFN-γ and HMGB-1 levels,serum ALT,AST,TB,BUN,Cr and CK-MB levels,MPO activity,PaCO2 and BE value were significantly increased,and WBC and Plt counts,pH value,PaO2 and survival rate within 72 h were decreased in MDP group (P ＜ 0.05).The pathologic changes of heart,liver,lung and kidney were obvious in S and MDP groups and severer in MDP group.Conclusion After a third-degree 20% total body surface area scald burn,MDP induces excessive production of inflammatory cytokines accompanying with multiple organ damage ; thus the model of sepsis is successfully established after scald burn in rats.

Objective To investigate the effect of electro-acupuncture at zusanli on Severe thermal injury-induced acute lung injury in rats.Methods Forty male SD rats weighing 200-250 g were used in this study.Thirty percent of the total body surface (TBS) was shaved chemically with 20% sodium sulfate and then exposed to 99-100℃ water for 12 s.The animals with third degree thermal injury involving 30% TBS were randomly divided into 5 groups(n=8 each):group Ⅰ control(group C);groupⅡ thermal injury;group Ⅲ electro-acupuncture at zusanli;group Ⅳ electric stimulation of non-acupoint and group Ⅴ electro-acupuncture at zusanli+α-bungarotoxin α-BGT).In group Ⅲ,Ⅳ,and Ⅴ electro-stimulation(3 v,2 ms,3 Hz) of zusanli or non-acupoint was performed for 12 min immediately after thermal injury model was established and every 8 h.hung specimens were obtained at 48 h after thermal injury for microscopic examination.The pulmonary HMGB-l protein level was measured by ELISA.The expression of HMGB-1 mRNA and protein in the lung was determined by RT-PCR and immuno-histochemistry respectively.Results Thermal injury induced leucocytosis in the interstitial capillaries,interstitial edema,intra-alveolar fibrin deposit,blebbing of type Ⅱ alveolar lining cells and decrease in lamellar body.Both expression of HMGB-1 mRNA and protein in the lung was significantly enhanced at 48 h after thermal injury.Electrical stimulation of zusanli significantly down-regalated the expression of HMGB-1 mRNA and protein in the lung.However,α-BGT pretreatment reversed the effects of electrical stimulation of zusanli.Conclusion Electrical stimulation of zusanli could significantly ameliorate severe thermal injury-induced acute lung injury through inhibition of HMGB-1 mRNA and protein expression and activation of cholinergic anti-inflammatory pathway mediated by nicotinic acetylcholine receptor α7 subunit.