Objective: To study human peripheral blood dendritic cells(DC) inducing Th0 differentiation to Th1/Th2 at different DC/Th0 ration。 Methods: Mononuclear cells in healthy human peripheral blood were induced to DC through tradition method,4 days of culture of mixed DC/Th0 different ration( 1∶4 and 1∶300),harvesting Th,after 48 h of CD3 mAb and CD28 mAb expantion,in supernant IL-4 and IFN-? measurement were determined using ELASA。 Results:Expresse CD83(73.4%), CD86 (90.3%), HLA-DR(68.5%),CD40(73.7%).DC/Th0 ration(1∶4) produced IFN-? (34?4.3) ?g/L, a little amount of IL- 4(25?4.3) ng/L. DC/Th0 ration(1∶300) produced IFN-?(3.5?1.2) ?g/L, a large amount of IL- 4(350?120) ng/L. Conclusion:Highly mixed DC/Th0 ration induced Th0 differentiation to Th1, low mixed DC/Th0 ration induced Th0 differentiation to Th2, DC regulating immunology response type were plastic which provided the basis for clinic GVHD therapy.

Objective To investigate the role of glycogen synthase kinase-3β (GSK-3β) in the trafficking of NR1-containing N-methyl-D-aspartate receptor (NMDAR) and NR2B-containing NMDAR in the spinal cord in rats with incisional pain (IP) and remifentanil-induced hyperalgesia.Methods Twenty-four male Sprague-Dawley rats,aged 2-3 months,weighing 240-260 g,were randomly divided into 3 groups ( n =8 each):control group (group C),remifentanil group (group R) and GSK-3β inhibitor TDZD-8 group (group TDZD-8).A 1-cm longitudinal incision was made in the plantar surface of the left hindpaw in anesthetized rats.In group C,dimethyl sulfoxide (DMSO) 2 ml/kg was injected and then the normal saline (equal to the volume of remifentanil) was infused for 60 min via the caudal vein.In group R,DMSO 2 ml/kg was injected before IP was made and then remifentanil was infused at a rate of 1.2 μg· kg- 1 ·min- 1 for 60 min.In group TDZD-8,TDZD-8 2 ml/kg was injected before IP was made and then remifentanil was infused at a rate of 1.2 μg·kg-1 ·min-1 for 60 min.Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured 24 h before infusion of normal saline or remifentanil and at 2,6,24 and 48 h after intravenous injection.The rats were sacrificed after the last measurement of the pain threshold and L4-6 segment of the spinal cord was removed to determine the expression of NMDAR NR1 and NR2B subunits in cell membrane (s) and cytoplasm (i) in the spinal cord by Western blot analysis.The ratios of sNR1/iNR1 and sNR2B/iNR2B were calculated.Results Compared with group C,PWT was significantly decreased and PWL was significantly shortened,the expression of sNR1 and sNR2B was up-regulated,the expression of iNR1 and iNR2B was down-regulated,and the ratios of sNR1/iNR1 and sNR2B/iNR2B were significantly increased in groups R and TDZD-8 (P ＜ 0.05).Compared with group R,PWT was significantly increased and PWL was significantly prolonged,the expression of sNR1 and sNR2B was down-regulated,the expression of iNR1 and iNR2B was up-regulated,and the ratios of sNR1/iNR1 and sNR2B/iNR2B were significantly decreased in group TDZD-8 ( P ＜ 0.05).Conclusion GSK-3β is involved in the regulation of trafficking of NR1-containing NMDAR and NR2B-containing NMDAR from cytoplasm to cell membrane in the spinal cord in rats with IP and hyperalgesia induced by remifentanil.

Objective To evaluate the relationship between delta opioid receptors (DORs) and activity of glycogen synthase kinase-3β (GSK-3β) in the spinal cord during hyperalgesia induced by remifentanil in a rat model of incisional pain (IP).Methods Twenty-four male Sprague-Dawley rats,aged 240-260 g,weighing 2-3 months,were randomly divided into 3 groups (n =8 each) using a random number table:control group (group C),remifentanil+IP group (group R+I) and DOR antagonist naltrindole group (group N).A 1-cm longitudinal incision was made in the plantar surface of the left hindpaw in anesthetized rats.In group C,the equal volume of normal saline was injected intraperitoneally,and normal saline was then infused for 60 min at the same rate.In group R+I,the equal volume of normal saline was injected intraperitoneally,remifentanil was infused for 60 min at 1.2 μg · kg-1 · min-1,and IP was produced immediately after onset of remifentanil infusion.In group N,naltrindole 0.1 mg/kg was injected intraperitoneally,remifentanil was infused for 60 min at 1.2 μg · kg-1 · min-1,and IP was produced immediately after onset of remifentanil infusion.At 24 h before infusion of normal saline or remifentanil and 2,6,24 and 48 h after iv administration (T0-4),the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were sacrificed after the last measurement of pain threshold,the lumbar segment (L4-6) of the spinal cord was removed for determination of the expression of GSK-3β,phosphor-GSK-3β (pGSK-3β) (by Western blot) and GSK-3β mRNA (realtime PCR).The ratio of pGSK-3β /GSK-3β was calculated.Results Compared with group C,the MWT was significantly decreased,and the TWL was shortened at T1 4,the expression of GSK-3β,pGSK-3β and GSK-3β mRNA was up-regulated,and pGSK-3β/GSK-3β ratio was decreased in R + I and N groups.Compared with group R + I,thc MWT was significantly incrcased,and the TWL was prolonged at T1-4,the expression of GSK-3β,pGSK-3β and GSK-3β rmRNA was down-regulated,and pGSK-3β/GSK-3β ratio was increased in group N.Conclusion Activation of DOR is involved in enhancement of activity of GSK-3β in the spinal cord during hyperalgesia induced by remifentanil in a rat model of IP.

γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

Objective To observe the effect of xinmailong(XML) injection on rat pulmonary hypertension(PH) by monocrotaline(MCT).Methods SD rats were given a single intraperitoneal injection of MCT with 60mg/kg to induce the mold of PH. Three weeks later,these rats were treated with XML for 8d,and then PH was measured using Sun Puo’s method.Results PH and right ventricle hypertrophy model had been set up successfully by MCT.The ratio of wet weight(wW) and dry weight(dW) of the lung increased with P

Objective To investigate the changes in total and surface N-methyl-D-aspartate receptor (NMDAR) NR1,NR2A and NR2B subunits in spinal cord in a rat model of incisional pain(IP)-remifentanil-induced hyperalgesia.Methods Thirty-two male SD rate in which caudal vein catheter were successfully placed were randomly divided into 4 groups ( n =8 each):control group(group C),remifentanil group(group R),IP group (group I) and IP + remifentanil group (group R + I).In groups R and C remifentanil 1.2 μg· kg-1 · min-1 or the same volume of normal saline was infused intravenously for60 main.In groups R + I and I,a 1 cm longitudinal incision was made in the plantar surface of left hindpaw and remifentanil 1.2 μg· kg-1 ·min-1 or the same volume of normal saline was infused intravenously for 60 min.Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured at 24 h before and 2,6,24,48 h after remifentanil or normal saline administration.The animals were sacrificed after last pain threshold measurement.The L4-6 segment of the spinal cord was isolated for determination of the expression of the total and surface NMDAR NR1,NR2A and NR2B subunits in spinal cord by Western blot.The ratio of surface NR2B/NR2A was calculated.Results Compared with group C,PWT was significantly decreased,PWL shortened,the expression of the total and surface NMDAR NR1,NR2A and NR2B subunits up-regulated and the ratio of surface NR2B/NR2A increased in groups 1,R and R + I( P ＜ 0.05).Compared with groups R and I,PWT was significantly decreased,PWL shortened,the expression of the total and surface NMDAR NR1,NR2A and NR2B subunits up-regulated and the ratio of surface NR2B/NR2A increased in group R + I( P ＜ 0.05).There was no significant difference in the total and surface NMDAR NR2A subunits among the four groups( P ＞ 0.05).Conclusion The up-regulation of total and surface NMDAR NR1 and NR2B subunits and the increased percentage of surface NR2B subunits in rats spinal cord may be involved in the development of incisional pain- remifentanil- induced hyperalgesia.

Objective To evaluate the changes in the expression of protein interacting with Cα kinase 1 (PICK1) in the spinal cord and dorsal root ganglion (DRG) neurons during remifentanil-induced hyperalgesia in rats with incisional pain.Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 42-49 days, were randomly divided into 4 groups (n =8 each) using a random number table: control group (group C) , incisional pain group (group Ⅰ) , remifentanil group (group R), and remifentanil + incisional pain group (group R + Ⅰ).In R and R+Ⅰ groups, remifentanil was infused intravenously for 60 min at the rate of 1.2 p,g · kg-1 · min-1.In C and Ⅰ groups, normal saline was infused intravenously for 60 min at the rate of 0.12 ml · kg-1 · min-1.In Ⅰ and R+Ⅰ groups, the model of incisional pain was established, and remifentanil and normal saline were infused intravenously, respectively, at the same time.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before normal saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of normal saline or remifentanil infusion (T1-4).The rats were sacrificed after the last measurement of pain threshold.The lumbar segment (L4-6) of the spinal cord and left DRGs were removed for determination of the expression of PICKl mRNA (by quantitative real-time reverse transcriptase-polymerase chain reaction) and PICK1 protein (by Western blot).Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in R and R+Ⅰ groups, and the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group Ⅰ (P＞0.05).Compared with group Ⅰ, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in group R+Ⅰ (P＜0.05).Compared with group R, the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group R+ Ⅰ (P＞0.05).Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to up-regulation of PICK1 expression in the spinal cord and DRG neurons of rats with incisional pain.

Objective To investigate the influence of dexmedetomidine on expressions of protein kinase c (PKC)γ, cal?cium/calmodulin-dependent protein kinase (CaMK)Ⅱαand pCaMKⅡαin spinal cord in rats with incisional pain (IP) and remifentanil-induced hyperalgesia. Methods Forty male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, were randomly divided into 5 groups (n=8 each):blank control group (group C), remifentanil+incisional pain group (group R+I), dexmedetomidine + remifentanil + incisional pain group (group D+R+I), dexmedetomidine + remifentanil + incisional pain+phorbol myristate acetate+DMSO group (group D+R+I+P+DMSO) and dexmedetomidine+remifentanil+incisional pain+DMSO group (group D+R+I+DMSO). The incisional pain rat model was established by a plantar incision in left hind paw. Remifentanil was infused at a rate of 1.2μg·kg-1·min-1 for 90 min via the caudal vein. Dexmedetomidine was adminis?tered subcutaneously at a dose of 50μg/kg at 30 min before plantar incision. Phorbol myristate acetate and DMSO were intra?thecally injected at a dose of 10 μL. Paw withdrawal latency (PWL) to thermal stimulation and paw withdrawal threshold (PWT) to von Frey hair stimulation were measured 24 h before remifentanil infusion (T0) and at 2, 6, 24 and 48 h (T1-4) after intraveonus remifentanil injection. The rats were sacrificed after the last behavioral test and the L 4-6 segment of spinal cord was removed to determine the expressions of PKCγ, CaMKⅡαand pCaMKⅡαin spinal cord by Western blot analysis. Re? sults Compared with group C, the value of PWL was significantly shortened and PWT was significantly decreased except T0, and the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere up-regulated in other groups. Compared with group R+I, the value of PWL was significantly prolonged and PWT was significantly increased, the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere down-regulated in group D+R+I and group D+R+I+DMSO. Compared with group D+R+I, the value of PWL was significantly shortened and PWT was significantly decreased, the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere up-regulated in group D+R+I+P+DMSO. Compared with group D+R+I+P+DMSO, the value of PWL was significantly prolonged and PWT was significantly increased, the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere down-regulated in group D+R+I+DMSO. Conclusion Dexmedetomidine can reduce the expressions of PKCγ, CaMKⅡαand pCaMKⅡαin spinal cord in rats with IP and hyperalgesia induced by remifentanil.

Objective To investigate the role of Nrf2 on hydrogen treatment for intestinal injury caused by severe sepsis.Methods 152 male ICR mice were randomly divided into four groups:sham operation group,hydrogen control group,sepsis group,and hydrogen treatment group,each n=38.Sepsis model was reproduced by cecal ligation and puncture (CLP).The mice in sham operation group and hydrogen control group did not receive CLP,and the operative procedure was the same as follows.The mice in hydrogen control group and hydrogen treatment group received 1-hour inhalation of 2％ hydrogen 1 hour and 6 hours after sham operation or CLP.Twenty animals in each group were selected and observed for 7-day survival rate.Eighteen animals in each group were selected and sacrificed at 6,12 and 24 hours after CLP.The intestinal tissues were obtained to determine the expression of Nrf2 and high mobility group B1 (HMGB1) protein by Western Blot,and the expression of Nrf2 mRNA by reverse transcription-polymerase chain reaction (RT-PCR).The middle portion of jejunum was obtained to evaluate the degree of septic injury by light microscope after hematoxylin and eosin (HE) staining.Results There was no statistical signifieance in variables between sham operation group and hydrogen control group.Compared with sham operation group,the 7-day survival rate was significantly decreased in sepsis group (0 vs.100％,P＜0.05); compared with sepsis group,the 7-day survival rate was significantly increased in hydrogen treatment group (55％ vs.0,P＜0.05).Compared with sham operation group,the expression of Nrf2 protein (gray value) and Nrf2 mRNA were up-regulated in sepsis group at 6,12 and 24 hours after CLP (Nrf2 protein 6 hours:1.973 ± 0.350 vs.1.000 ± 0.000,t=4.411,P=0.002; 12 hours:2.367 ± 0.186 vs.1.000 ±0.000,t=10.210,P=0.000; 24 hours:2.517 ±0.280 vs.1.000 ±0.000,t=9.521,P=0.000; Nrf2 mRNA 6 hours:1.606 ± 0.271 vs.1.000 ± 0.000,t=3.631,P=0.002; 12 hours:1.692 ± 0.399 vs.1.000 ± 0.000,t=3.233,P=0.005; 24 hours:1.784 ± 0.341 vs.1.000 ± 0.000,t=3.894,P=0.001),and it was also the expression of HMGB1 (gray value) at 24 hours after CLP operation (1.507 ± 0.220 vs.1.000 ± 0.000,t=3.948,P=0.004).Compared with sepsis group,the expression of Nrf2 protein and Nrf2 mRNA in intestines were up-regulated at 6,12 and 24 hours after CLP in hydrogen treatment group (Nrf2 protein 6 hours:2.583 ± 0.395 vs.1.973 ± 0.350,t=2.765,P=0.024; 12 hours:2.725 ± 0.235 vs.2.367 ± 0.186,t=2.674,P=0.028; 24 hours:2.930 ± 0.212 vs.2.517 ± 0.280,t=2.595,P=0.032; Nrf2 mRNA 6 hours:2.008 ± 0.400 vs.1.606 ± 0.271,t=2.405,P=0.029; 12 hours:2.188 ± 0.475 vs.1.692 ±0.399,t=2.317,P=0.034; 24 hours:2.333 ±0.406 vs.1.784 ±0.341,t=2.728,P=0.015).Compared with sepsis group,the expression of HMGB1 was down-regulated significantly at 24 hours after CLP in hydrogen treatment group (1.147 ± 0.152 vs.1.507 ± 0.220,t=2.805,P=0.023).HE staining showed that there was significantly aggravated intestinal pathological injury in the mice of sepsis group; compared with sepsis group,the pathology was significantly less marked in hydrogen treatment group.Conclusion Through activation of Nrf2-antioxidant response element (ARE) pathway,hydrogen may increase the level of Nrf2,which is a kind of protective protein,in the intestine of mice,thus decreases the level of late pro-inflammatory factor,HMGB1,and it may protect the intestinal tissues in septic mice and increase the survival rate significantly.

Objective To evaluate the effects of hydrogen (H2) on nuclear factorE2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway in lung tissues in septic mice.Methods Seventy-two male ICR mice,weighing 20-25 g,aged 6 weeks,were randomly divided into 4 groups (n =18 each) using a random number table:sham operation group (group SH),group H2,sepsis group (group S),and sepsis + H2 group (group S + H2).Sepsis was produced by cecal ligation and puncture (CLP).H2 and S + H2 groups inhaled 2％ H2 for 1 h starting from 1 and 6 h after CLP.Six mice in each group were chosen and sacrificed at 7,12 and 24 h after CLP (T1-3).The pulmonary specimens were obtained to determine the expression of Nrf2 and HO-1 protein (by Western blot) and Nrf2 mRNA (by RT-PCR).At 24 h after CLP,the pathological changes of lungs were scored,wet/dry lung weight ratio (W/D ratio) was determined,and the expression of high mobility group box-1 (HMGB-1) in lung tissues was measured (by Western blot).Results Compared with group SH,the pathological scores and W/D ratio were significantly increased,and the expression of Nrf2 protein and mRNA,HO-1 and HMGB1 was up-regulated in S and S + H2 groups,while no significant change was found in the indexes mentioned above in group H2.Compared with group S,the pathological scores and W/D ratio were significantly decreased,the expression of Nrf2 protein and mRNA and HO-1 was up-regulated and HMGB1 expression was down-regulated in group S + H2.Conclusion The mechanism by which H2 reduces acute lung injury in septic mice is related to activation of Nrf2/ARE pathway.