Role of ERK1/2/CREB/BDNF signaling pathway in dexmedetomidine-induced inhibition of propofol-caused apoptosis in isolated hippocampal neurons of fetal rats / 中华麻醉学杂志

Objective:To evaluate the role of extracellular signal-regulated kinase 1/2 (ERK1/2)/cyclic adenosine monophosphate response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signaling pathway in dexmedetomidine-induced inhibition of propofol-caused apoptosis in isolated hippocampal neurons of fetal rats.Methods:Pregnant Sprague-Dawley rats at 16 days of gestation were sacrificed, and the fetal rats were taken out, and hippocampal neurons of fetal rats were obtained and primarily cultured in vitro for 7 days.The neurons were divided into 9 groups ( n=12 each) using a random number table method: control group (group C), fat emulsion group (group I), dimethyl sulfoxide (DMSO) group, dexmedetomidine group (group D), propofol group (group P), propofol plus dexmedetomidine group (group PD), PD98059 plus propofol plus dexmedetomidine group (group PDP), MH89 plus propofol plus dexmedetomidine group (group HDP), and KG501 plus propofol plus dexmedetomidine group (group KDP). Group C received no treatment.In group I, 20% fat emulsion was added, and the neurons were incubated for 30 min, and 0.25% DMSO was added in group DMSO, and the neurons were incubated for 30 min.Dexmedetomidine at a final concentration of 10 μmol/L was added, and the neurons were incubated for 30 min in group D. Propofol at a final concentration of 100 μmol/L was added, and the neurons were incubated for 3 h in group P. In group PD, dexmedetomidine at a final concentration of 10 μmol/L was added, the neurons were incubated for 30 min, propofol at a final concentration of 100 μmol/L was added, and the neurons were incubated for 3 h. In PDP, HDP and KDP groups, 25 μmol PD98059 (p-ERK1/2 inhibitor), 10 μmol H89 (p-CREB inhibitor) and 25 μmol KG501 (CREB inhibitor) were added, respectively, the neurons were incubated for 30 min, dexmedetomidine at a final concentration of 10 μmol/L was added, the neurons were incubated for 30 min, and propofol at a final concentration of 100 μmol/L was added, and the neurons were incubated for 3 h. The cell ultrastructure was observed with the transmission electron microscope, the apoptosis in neurons was detected by flow cytometry, the expression of ERK1/2, CREB and BDNF mRNA was detected by quantitative real-time polymerase chain reaction, and the expression of p-ERK1/2, CREB, p-CREB, BDNF and cleaved caspase-3 was detected by Western blot. Results:Compared with group C, the apoptosis rate was significantly increased, the expression of p-ERK1/2 and p-CREB was down-regulated, and the expression of cleaved caspase-3 was up-regulated in P, PD, PDP, HDP and KDP groups, and the expression of BDNF was significantly down-regulated in P, PDP, HDP and KDP groups ( P<0.05). Compared with group P, the apoptosis rate was significantly decreased, the expression of p-ERK1/2, p-CREB and BDNF was up-regulated, and the expression of cleaved caspase-3 was down-regulated in group PD ( P<0.05). Compared with group PD, the apoptosis rate was significantly increased, the expression of p-ERK1/2, p-CREB and BDNF was down-regulated, and the expression of cleaved caspase-3 was up-regulated in PDP, HDP and KDP groups ( P<0.05). Conclusion:The ERK1/2/CREB/BDNF signaling pathway is involved in dexmedetomidine-induced inhibition of propofol-caused apoptosis in isolated hippocampal neurons of fetal rats.

Effects of intrathecal injection Roscovitine on the pain behavior and the expression of ERK1/2 and PPARγ protein in spinal cord in rats with neuropathic pain / 中华行为医学与脑科学杂志

Objective To investigate the effects of intrathecal injection of Roscovitine on the pain behavior and the expression of pERK1/2 and pPPARγ receptor in spinal cord in rats with neuropathic pain.Methods Fifty-two male adult Sprague-Dawley rats of 220～ 250 g were randomly divided into 4 groups:Sham+DMSO group (dimethyl sulfoxide,S+D group),Sham+Roscovitine (S+R) group,CCI+ DMSO group (I+D),CCI+ Roscovitine (I+R) group.The corresponding drugs were administered by intrathecal injection from the seventh day after CCI once a day for 14 days.The right hind paw mechanical threshold value (PMWT) was measured by Von Frey filament and paw thermal withdrawal latency(PWTL) was measured by Hargreaves methods before 1 day and 3 d,7 d,10 d,14 d after surgery.The spinal cord lumbar enlargement was taken at 14 d after surgery,and Cdk5,p35,phosphorylated ERK protein (pERK),total ERK protein (ERK),phosphorylated PPARγprotein (pPPARγ) and total PPARγ protein (PPARγ) were detecteded by Western blot.Results Roscovitine was administered by intrathecal injection alleviated mechanical allodynia and thermal hyperalgesia of CCI rats.Compared with I+D group,PWMT in I+R group at 7 d,10 d,14 d after administration((4.65±0.08)g,(6.47±0.12)g,(7.90±0.19)g,) vs ((3.71 ±0.06)g,(2.45±0.17)g,(2.31±0.15)g) (Fgroup =505.71,P＜0.05,Ftime×group =15.33,P＜0.05),PWTL((9.22±0.33) s,(13.52±0.43) s,(12.63±0.88) s) vs ((8.02±0.20) s,(5.90±0.28) s,(5.40±0.38) s) (Fgroup =355,P＜0.05,F time×group =8.589,P＜0.05) were significantly increased.Compared with I+D group(p35 (0.58±0.02),pERK (1.12±0.13),pPPARγ (0.77±0.16)),p35 (0.46±0.04,F=11.06,P＜0.05) and pERK(0.79±0.11,F =22.91,P＜ 0.05) in I+ R group,were significantly dereased,and the expression of pPPARγ(0.99±0.13,F =17.62,P＜0.05))was significantly increased.Conclusion Intrathecal injection Roscovitine can alleviate both mechanical allodynia and thermal hyperalgesia in rats with neuropathic pain,and its mechanisms may be related to the inhibition of Cdk5/p35 and ERK activity,enhancement of PPARγ activity in the spinal dorsal horn.

Role of cAMP-PKA-CREB signaling pathway in hypoxic preconditioning-induced reduction of propofol-induced central neurotoxicity in developing rats / 中华麻醉学杂志

Objective To evaluate the role of cyclic adenosine monophosphate-protein kinase A-cAMP response element-binding protein (cAMP-PKA-CREB) signaling pathway in hypoxic preconditioninginduced reduction of propofol-induced central neurotoxicity in the developing rats.Methods A total of 70 SPF male Sprague-Dawley rats,aged 7 days,weighing 10-15 g,were divided into 7 groups (n=10 each) using a random number table method:normal saline group (N group),propofol group (P group),hypoxic preconditioning plus propofol group (HP group),hypoxic preconditioning plus propofol plus PKA inhibitor H89 group (HPH group),propofol plus PKA agonist SP-CAMP group (PS group),normal saline injected via the lateral cerebral ventricle group (NI group),and 5％ dimethyl sulfoxide (DMSO) injected via the lateral cerebral ventricle group (DI group).In P group,propofol 50 mg/kg was intraperitoneally injected,and an increment of propofol 50 mg/kg was given after recovery of righting reflex.The equal volume of normal saline was given instead in N group.In HP group,hypoxic preconditioning (rats were subjected to 5 cycles of 10-min hypoxia of 8％ O2 and 1O-min normoxia of 21％ O2) was performed,and propofol was intraperitoneally injected at 2 h after the end of hypoxic preconditioning and the method was similar to those previously described in P group.In HPH group,H89 5 μmol/5 μl was injected via the lateral cerebral ventricle,and 30 min later the other treatment was similar to those previously described in HP group.In PS group,SP-CAMP 20 nmol/5 μl was injected via the lateral cerebral ventricle,and 30 min later propofol was injected using the method previously described in P group.In NI and DI groups,5 μl normal saline and 5％ DMSO were injected via the lateral cerebral ventricle,respectively.Rats were immediately sacrificed after the righting reflex was recovered,brains were removed and hippocampi were isolated and cut into sections which were stained with haematoxylin and eosin for determination of PKAc and p-CREB positive cells (by i mmuno-histochemistry) and expression of cleaved caspase-3,Bcl-2,Bax,PKAc and posphorylated (p-CREB) protein (by Western blot).Results Compared with N group,the expression of cleaved caspase-3 and Bax was significantly up-regulated,the expression of Bcl-2,PKAc and p-CREB was downregulated,and the percentage of PKAc and p-CREB positive cells was decreased (P＜0.05),hippocampal cells had irregular arrangement,and cells was atrophied in P group.Compared with P group,the expression of cleaved caspase-3 was significantly down-regulated,the expression of Bcl-2,PKAc and p-CREB was up-regulated,and the percentage of PKAc and p-CREB positive cells was increased in HP and PS groups,and the expression of Bax was down-regulated (P＜0.05),the hippocampal cells were arranged neatly,the cytoplasm was abundant,and the nuclei were visible in HP group.Compared with HP group,the expression of cleaved caspase-3 was significantly up-regulated,the expression of Bcl-2,PKAc and p-CREB protein was down-regulated and the percentage of PKAc and p-CREB positive cells was decreased (P＜0.05),the cells had irregular arrangement and shrinked,and nuclear condensation was found in cells in HPH group.Conclusion The mechanism by which hypoxic preconditioning reduces propofol-induced central neurotoxicity may be related to activating cAMP-PKA-CREB signaling pathway in the developing rats.

Long-term effects of dexmedetomidine on the brain development in propofol-induced neonatal rats / 临床麻醉学杂志

Objective To explore the long-term effects of dexmedetomidine on the brain development in propofol-induced neonatal rats.Methods Thirty-five seven-day-old Sprague-Dawley rats of both genders,weighing 10-15 g,were randomly divided into seven groups (n =5) using a random number table:normal saline group (group N),intralipid group (group F),propofol 100 mg/kg group (group P),dexmedetomidine 75 μg/kg (group D),dexmedetomidine 25 μg/kg,50 μg/kg and 75μg/kg+propofol 100 mg/kg groups (groups PD25,PD50 and PD75),neonatal rats in each group were treated according to the corresponding dosing regimen.After fully awake,the rats were allowed to mature until postnatal week 9 and the spatial learning and memory capacities were tested by Morris water maze.The rats were sacrificed after the tests.Brain was sliced for determination of hippocampal apoptosis by TUNEL assays and the expression of postsynaptic density protein 95 (PSD95) by immunohistochemistry.Results Compared with group N,the escape latency was significantly prolonged,the times of platform crossing were significantly decreased,the hippocampal apoptosis ratio was significantly increased and the expression of PSD95 was significantly down-regulated in groups P,PD25 and PD50 (P＜0.05).Compared with group P,the escape latency was significantly shortened,the times of platform crossing were significantly increased and the hippocampal apoptosis were significantly decreased in groups PD50 and PD75 (P＜0.05),the expression of PSD95 was up-regulated in group PD75 (P＜0.05).Compared with group PD25,the escape latency was significantly shortened,the number of platform crossing was significantly increased and the hippocampal apoptosis were significantly decreased in groups PD50 and PD75 (P＜0.05),the expression of PSD95 was significantly up-regulated in group PD75 (P＜0.05).Compared with group PD50,the hippocampal apoptosis were significantly decreased,the expression of PSD95 was significantly up-regulated in group PD75 (P＜ 0.05).Conclusion The addition of dexmedetomidine 50,75 μg/kg attenuates propofol-induced neurocognitive impairment in neonatal rats after aducthood,partially by attenuating hippocampal apoptosis and upregulating the expression of PSD95.Dexmedetomidine alone was not neurotoxic to the developing brain.

Role of hippocampal PI3K/Akt/GSK-3β signaling pathway in dexmedetomidine-induced reduction of long-term cognitive decline caused by propofol in neonatal rats / 中华麻醉学杂志

Objective To evaluate the role of hippocampal phosphatidylinositol 3-kinase/serinethreonine kinase/glycogen synthase kinase-3 beta (PI3K/Akt/GSK-3β) signaling pathway in dexmedetomidine-induced reduction of long-term cognitive decline caused by propofol in neonatal rats.Methods A total of 110 clean healthy male Sprague-Dawley rats,aged 7 days,weighing 10-15 g,were divided into 11 groups (n=10 each) using a random number table:normal saline group (NS group),fat emulsion group (F group),10％ dimethyl sulfoxide (DMSO) group (D2 group),dexmedetomidine group (DEX group),TDZD-8 group (TDZD group),10％ DMSO group (D1 group),LY294002 group (LY group),propofol group (P group),dexmedetomidine plus propofol group (PD group),LY294002 plus dexmedetomidine plus propofol group (LYPD group) and TDZD-8 plus dexmedetomidine plus propofol group (TDPD group).Normal saline,fat emulsion,10％ DMSO 100 μl,dexmedetomidine 75 μg/kg and TDZD-8 1 mg/kg were intraperitoneally injected in NS,F,D2,DEX and TDZD groups,respectively.10％ DMSO 5 μ1 and LY294002 25 μg/5 μl were injected via the lateral cerebral ventricle in D1 and LY groups,respectively.Propofol 50 mg/kg was intraperitoneally injected,and an increment of propofol 50 mg/kg was given after recovery of righting reflex in group P.Dexmedetomidine 75 μg/kg was intraperitoneally injected and 30 min later propofol was injected in group PD.LY294002 was injected,30 min later dexmedetomidine was injected,and 30 min later propofol was injected in group LYPD.In group TDPD,TDZD-8 was injected and the other treatment was similar to those previously described in group LYPD.Then the rats were fed to 9 weeks old after returning to the state of consciousness.Morris water maze test was performed to evaluate the cognitive function.Rats were then sacrificed and their hippocampi were harvested for detection of the expression of PI3K,Akt,GSK-3β and PSD95 mRNA (using real-time polymerase chain reaction) and expression of Akt,pAkt(ser473),GSK-3β,pGSK-3β(ser9) and PSD95 (by Western blot).Results Compared with NS group,the escape latency was significantly prolonged,the number of crossing the original platform was reduced,the expression of PI3K,Akt and PSD95 mRNA was down-regulated,the expression of GSK-3β mRNA was up-regulated,p-Akt(ser473)/Akt ratio was decreased,the expression of PSD95 was downregulated,and pGSK-3β (ser9)/GSK-3β ratio was increased in P group (P＜ 0.05).Compared with P group,the escape latency was significantly shortened and the number of crossing the original platform was increased in PD,LYPD and TDPD groups,the expression of PI3K,Akt and PSD95 mRNA was up-regulated,and the expression of GSK-3β mRNA was down-regulated in PD group,and pAkt(ser473)/Akt ratio was increased,the expression of PSD95 was up-regulated,and pGSK-3β (ser9)/GSK-3β ratio was decreased in PD and TDPD groups(P＜0.05).Compared with PD group,the escape latency was significantly prolonged,the number of crossing the original platform was reduced,the expression of GSK-3β mRNA was up-regulated,the expression of PSD95 mRNA was down-regulated,pAkt (ser473)/Akt ratio was decreased,the expression of PSD95 was down-regulated,and pGSK-3β (ser9)/GSK-3β ratio was increased in LYPD group,and the escape latency was significantly shortened,the number of crossing the original platform was increased,the expression of GSK-3β mRNA was down-regulated,the expression of PSD95 mRNA was up-regulated,pGSK-3β(ser9)/GSK-3β ratio was decreased,and the expression of PSD95 was up-regulated in TDPD group(P＜0.05).Conclusion Hippocampal PI3K/Akt/GSK-3β signaling pathway is involved in dexmedetomidine-induced reduction of long-term cognitive decline caused by propofol in neonatal rats.

Effect of dexmedetomidine on activity of GSK-3β during propofol-induced apoptosis in hippocampal nerve cells of newborn rats / 中华麻醉学杂志

Objective To evaluate the effect of dexmedetomidine on the activity of glycogen synthase kinase-3 beta (GSK-3β) during propofol-induced apoptosis in hippocampal nerve cells of newborn rats.Methods Sixty male 7-day-old Sprague-Dawley rats,weighing 10-15 g,were divided into 6 groups (n=10 each) using a random number table:normal saline group (group NS),fat emulsion group (group F),propofol group (group P) and different doses of dexmedetomindine groups (group D25,group D50 and group D75).Normal saline and fat emulsion 100 μl were injected intraperitoneally in group NS and group F,respectively.In group P,propofol 50 mg/kg was intraperitoneally injected,and an increment of propofol 50 mg/kg was added after righting reflex completely recovered,with the total amount of 100 mg/kg.In group D25,group D50 and group D75,dexmedetomidine 25,50 and 75 μg/kg were intraperitoneally injected,respectively,and 30 min later propofol 100 mg/kg was administered.At 2 h after emergence,the rats were sacrificed,their brains were removed for determination of apoptosis in hippocampal nerve cells (by TUNEL),and the hippocampi were isolated for detection of the expression of GSK-3β and phosphorylated GSK-3β (p-GSK-3β) by Western blot analysis.The apoptosis index (AI) and ratio of p-GSK-3β/GSK-3β were calculated.Results Compared with group NS,AI was significantly increased,the expression of p-GSK-3β was down-regulated,and the p-GSK-3β/GSK-3β ratio was decreased in P,D25,D50 and D75 groups (P＜0.05).Compared with group P,AI was significantly decreased,the expression of p-GSK-3β was up-regulated,and the p-GSK-3β/GSK-3β ratio was increased in D25,D50 and D75 groups (P＜0.05).Compared with group D25,AI was significantly decreased (P＜0.05),and no significant change was found in the expression of p-GSK-3β or ratio of p-GSK-3β/GSK-3β in D50 and D75 groups (P＞0.05).Compared with group D50,AI was significantly decreased (P＜0.05),and no significant change was found in the expression of p-GSK-3β or ratio of p-GSK-3β/GSK-3β in group D75 (P＞0.05).Conclusion The mechanism by which dexmedetomidine attenuates propofol-induced apoptosis in hippocampal nerve cells may be related to inhibition of GSK-3β activity in newborn rats.

Effect of naloxone in combination with morphine on the growth of subcutaneous tumor of human gastric cancer MGC-803 cells in nude mice / 临床麻醉学杂志

Objective To observe the effects of naloxone in combination with morphine on the growth of subcutaneous tumor of human gastric cancer MGC-803 cells in nude mice.Methods The model of subcutaneous tumor of human gastric cancer MGC-803 cells in nude mice was established.Fifty nude mice were randomly divided into 5 groups: control group (group C),normal saline group (group S),20 mg/kg morphine group (group M),and 1 mg/kg naloxone group (group N),and 1 mg/kg naloxone+20 mg/kg morphine group (group NM).The mice in group C received no treatment,while the mice in group S,group M,group N and group NM were injected with 1.5 ml/kg saline,20 mg/kg morphine,1 mg/kg naloxone,and 1 mg/kg naloxone+20 mg/kg morphine per day,respectively.The caliper was used to measure the tumor sizes every the other day.The mice in each group received intraperitoneal injection of the drugs for 2 week.Then the relative volume (RTV) of tumor was calculated.The expression of Cyclin D1,VEGF,MMP-9 mRNA and proteins were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR),immunochemistry staining and Western blot.Results RTV in group M (2.21±0.62)% was significantly lower than that in group C (3.16±0.68)%,group S (2.98±0.61)%,group N (3.16±0.35)% and group NM (2.64±0.37)% (P<0.05).RTV in group NM was significantly lower than that in group C,group S and group N (P<0.05).Compared with group C,the expression of Cyclin D1,VEGF,and MMP-9 in group M were significantly decreased (P<0.05).The organizational structure of the subcutaneous tumor in groups C,S,N and NM was almost normal.Cytoplasm vacuolization,disruption of nuclear membrane and chromatin margination were occured in group M.While the level of Cyclin D1,VEGF,and MMP-9 in group NM was increased compared to group M (P<0.05).Conclusion Morphine could inhibit the growth of subcutaneous tumor of human gastric cancer MGC-803 cells in nude mice by downregulating the expression of Cyclin D1,VEGF,and MMP-9.Naloxone could antagonize the anti-growth effects of morphine.

Effects of dexmedetomidine on PI3K/Akt pathway in hippocampus of propofol anesthetized neonatal rats / 临床麻醉学杂志

Objective To explore the effect of dexmedetomidine on phosphoinositide 3-kinase/protein kinase B (PI3K/Akt ) pathway in hippocampus of propofol anesthetized neonatal rats. Methods Eighty Sprague-Dawley male rats,aged 7 days,weighing 10-1 5 g,were randomly divided into 8 groups (n= 10 each):normal saline group (group N),DMSO group (group D),intralipid group (group I),propofol group (group P),dexmedetomidine 25 μg/kg,50 μg/kg and 75 μg/kg +propofol 100 mg/kg groups (groups PD25 ,PD50 and PD7 5 ),LY294002 25 μg + dexmedetomidine 75μg/kg + propofol 100 mg/kg group (group LYPD).The hippocampus of rats in all groups were taken 2 h after the animals fully awake.The ultrastructure of hippocampal neurons was observed by transmission electron microscope.The pAkt-(ser473 )protein and Akt protein in the hippocampus were evaluated by Western blot analysis.Results There was no significant difference in the expression of Akt protein among the eight groups.Compared with group N,the expression of pAkt (ser473)protein was significantly down-regulated in groups P,PD25 ,PD50 ,PD7 5 and LYPD (P <0.05).Compared with group P,the expression of pAkt (ser473)protein was increased significantly in groups PD7 5 and LYPD (P <0.05).Compared with group PD7 5 ,the expression of pAkt (ser473) protein was significantly down-regulated in group LYPD (P <0.05 ).The structure of hippocampal neurons was normal in groups N,I and D.Nuclear nuclei swelling,chromatin decreasing and mito-chondrion vacuolar degeneration were observed in group P while improved gradually with dexmedeto-midine in a dose-dependent manner in groups PD25 ,PD50 and PD7 5 .Neurons karyopyknosis,partial dissolution of nuclear membrane,chromatin condensation,mitochondria vacuolar degeneration were observed in group LYPD.Conclusion Dexmedetomidine pretreatment provides neuroprotection against propofol-induced hippocampal destruction by preserving PI3K/Akt pathway activity in the de-veloping brains.

Effect of morphine on growth of subcutaneous tumor of human gastric cancer cells in nude mice / 中华麻醉学杂志

Objective To evaluate the effect of morphine on the growth of subcutaneous tumor of human gastric cancer cells in nude mice.Methods Thirty SPF male BALB/C nude mice,aged 4-5 weeks,weighing 15-20 g,in which the model of subcutaneous tumor of human gastric cancer cell line MGC-803 was established,were randomly divided into 3 groups (n=10 each) using a random number table:control group (group C),normal saline group (group N),and morphine group (group M).The mice in group C received no treatment.Morphine 20 mg/kg was intraperitoneally injected once a day for 14 consecutive days in group M,while normal saline 1.5 ml/kg was given instead of morphine in group N.The caliper was used to measure the tumor size every 2 days starting from 3 days after beginning of administration,and the relative tumor volume was calculated.The nude mice were sacrificed on 15th day,and the tumor tissues were obtained for determination of nuclear factor-kappa B activity and Bcl-2 and Bax protein and mRNA expression by real-time reverse transcriptase polymerase chain reaction and Western blot.Results Compared with group C,the relative tumor volume was significantly decreased,the activity of nuclear factor-kappa B in tumor tissues was significantly decreased,the expression of Bcl-2 protein and mRNA was significantly down-regulated,and the expression of Bax protein and mRNA was significantly up-regulated at each time point in group M (P＜0.05),and no significant change was found in each parameter mentioned above in group N (P＞0.05).Conclusion Morphine can inhibit the growth of subcutaneous tumor of human gastric cancer cells in nude mice,and the mechanism is associated with promotion of apoptosis in tumor cells.

Effect of fentanyl on expression of VEGF-A and MMP-9 in subcutaneous tumor of human gastric cancer in nude mice / 中华麻醉学杂志

Objective To evaluate the effect of fentanyl on the expression of vascular endothelial growth factor A (VEGF-A) and matrix metallopeptidase-9 (MMP-9) in the subcutaneous tumor of human gastric cancer in nude mice.Methods Thirty SPF male BALB/C nude mice,aged 4-5 weeks,weighing 15-20 g,in which the model of subcutaneous tumor of human gastric cancer cell line MGC-803 was established,were randomly divided into 6 groups (n =5 each) using a random number table:control group (C group),normal saline group (NS group) and fentanyl 0.05,0.10,0.20 and 0.40 mg/kg groups (F1-4 groups).The mice in group C received no treatment.Fentanyl 0.05,0.10,0.20 and 0.40 mg/kg were intraperitoneally injected once a day for 14 consecutive days in F1 4 groups,respectively,while the equal volume of normal saline 1.5 ml/kg was given instead of fentanyl in group NS.The nude mice were sacrificed on 1 day after the end of administration,and the tumor tissues were obtained for examination of the ultrastructure of subcutaneous tumor (with a transmission electron microscope) and for detection of the expression of VEGF-A and MMP-9 (by immunohistochemistry and Western blot) and expression of VEGF-A and MMP-9 mRNA (by real-time reverse transcriptase polymerase chain reaction).Results No abnormality in the morphology of the subcutaneous tumor cells was observed in C and NS groups.The swollen nucleus,chromatin margination,nuclear fragmentation and apoptotic bodies were found in the subcutaneous tumor cells in F1-4 groups.Compared with group C,the expression of VEGF-A and MMP-9 protein and mRNA was significantly down-regulated in F1-4 groups (P＜0.05),and no significant change was found in each parameter mentioned above in group NS (P＞0.05).There was no significant difference in the expression of VEGF-A and MMP-9 protein and mRNA among F1-4 groups (P＞0.05).Conclusion The mechanism by which fentanyl inhibits the growth and metastasis of subcutaneous tumor cells of human gastric cancer is related to down-regulation of VEGF-A and MMP-9 expression in nude mice.

Effect of dexmedetomidine on expression of NGF in isolated hippocampal neurons of fetal rats incubated with propofol / 中华麻醉学杂志

Objective To evaluate the effect of dexmedetomidine on the expression of nerve growth factor (NGF) in isolated hippocampal neurons of fetal rats incubated with propofol.Methods Hippocampal neurons derived from the fetal rats of pregnant Sprague-Dawley rats at 5-13 days of gestation were primarily cultured for 7 days,and were inoculated in the culture plate at a density of 5×105 cells/ml.The neurons were randomly divided into 3 groups (n =15 each) using a random number table:control group (group C),propofol group (group P),and dexmedetomidine + propofol group (group DP).In group P,propofol with the final concentration of 100 μmol/L was added to the culture medium,and the cells were incubated for 3 h.In group DP,dexmedetomidine with the final concentration of 1 μmol/L was added to the culture medium,the cells were incubated for 30 min,and then propofol with the final concentration of 100 μmol/L was added to the culture medium,and the cells were incubated for 3 h.The viability of hippocampal neurons was assessed by CCK-8 assay.NGF mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction.NGF protein expression was detected by Western blot.Results Compared with group C,the viability of hippocampal neurons was significantly decreased,and the expression of NGF protein and mRNA was down-regulated in group P (P＜0.05).Compared with group P,the viability of hippocampal neurons was significantly increased,and the expression of NGF protein and mRNA was up-regulated in group DP (P＜0.05).Conclusion Dexmedetomidine improve propofol-induced decrease in the viability of isolated hippocampal neurons of fetal rats through up-regulating the expression of NGF.

Expression of microRNA-1284 in gastric cancer and underlying mecha-nism / 中国病理生理杂志

AIM:To evaluate the correlation between microRNA-1284 (miR-1284) and gastric cancer, and to investigate the underlying mechanism.METHODS: The expression of miR-1284 was examined by real-time PCR in 63 gastric cancer ( GC) tissue samples and 63 non-malignant adjacent tissue samples.The correlation between miR-1284 and the clinicopathological feature of GC was analyzed.Lentiviral vector containing miR-1284 was constructed and transfected into GC SGC-7901 cells.After transfection, the expression of miR-1284 was examined by real-time PCR.The cell activity was evaluated by CCK-8 assay.The cell cycle and apoptosis were determined by flow cytometry.The ability of cell migra-tion was measured by wound-healing assay.The potential target gene of miR-1284 was predicted by online bioinformatic softwares.The expression of JAG1 mRNA was examined by real-time PCR.The protein levels of JAG1, Notch1 and NF-κB were analyzed by Western blotting.RESULTS:Compared with non-malignant adjacent tissue samples, the results of real-time PCR showed significant downregulation of miR-1284 in 42 GC tissue samples ( P0.05), but significantly associated with histologic grading (P<0.05).Compared with LV-NC-GFP group and control group, after transfection of miR-1284 in LV-miR-1284 group, the expression of miR-1284 was significantly in-creased (P<0.05), the percentages of apoptotic cells and the cells in G0/G1 phase were significantly increased (P<0.05), the cells activity and ability of migration were significantly decreased (P<0.05), and the expression of JAG1, Notch1 and NF-κB was significantly decreased (P<0.05).CONCLUSION:The inhibitory effect of miR-1284 on gastric cancer may be associated with the regulation of its targeting gene JAG1.

Pathogenesis of complicated acute lung injury after percutaneous coronary intervention / 心血管康复医学杂志

Objective:To explore causes of complicated acute lung injury (ALI) after percutaneous coronary interven-tion (PCI) .Methods:According to blood gas analysis and chest imaging examination ,a total of 175 patients under-going PCI were divided into ALI group (n=62) and non-ALI group (n=113) .High performance liquid chromatog-raphy was used to measure plasma concentrations of epinephrine (E) and norepinephrine (NE) during perioperative period .Plasma levels of interleukin-6 (IL-6) and procalcitonin (PCT) and chest CT imaging changes were meas-ured .Fluorescent immunoassay was used to measure plasma level of brain natriuretic peptide (BNP) to assess impact of cardiac function on ALI .Results:Compared with non-ALI group on 1d after PCI ,there were significant rise in plasma levels of NE [ (2.51 ± 0.31) nmol/L vs .(6.91 ± 0.39) nmol/L] and E [ (1.23 ± 0.11) nmol/L vs .(6.03 ± 0.37) nmol/L] ,P<0.01 all;and significant rise in plasma levels of IL-6 [ (119.81 ± 17.23) pg/ml vs .(252.28 ± 34.23) pg/ml] ,PCT [ (0.88 ± 0.01) pg/ml vs .(4.99 ± 0.87) pg/ml] and BNP [ (927.82 ± 89.72) pg/ml vs . (3936.55 ± 131.78) pg/ml] in ALI group (P<0.01 all) .Chest CT indicated that lung tissue inflammation was seri-ous .Conclusion:In patients undergoing percutaneous coronary intervention ,complicated acute lung injury is related to hyperactive sympathetic activity ,postoperative inflammation and heart function status etc .

The effect of miR-1284 over-expression on gene expression proifling and invasion/metastasis of human gastric cancer SGC-7901 cells / 中国癌症杂志

Background and purpose:It has beenreported that miR-1284 is associated with gastric cancer lymph node metastasis in the research of microRNA microarray in human gastric cancer tissues. But the specific role of miR-1284 in gastric cancer has not been reported. The aim of this study was to investigate the effect of miR-1284 over-expression on the gene expression profiling and invasion/metastasis of human gastric cancer SGC-7901 cells. Methods:Gastric cancer SGC-7901 cells of LV-miR-1284 group were transfected with lentiviral vectors of miR-1284, cells of LV-NC-GFP group were transfected with lentiviral vectors without miR-1284, and cells of control group were not transfected with lentiviral vectors. The expression of miR-1284 was detected by the real-time fluorescent quantitative PCR. Differential expression genes were detected by the microRNA chip. Target genes of miR-1284 were predicted by the bioinformatics. Invasive ability was detected by the Transwell invasion assay. Metastasis ability was detected by subcutane-ously transplanted tumor model of nude mice.Results:Compared with LV-NC-GFP and control groups, the expressions of miR-1284 and 20 genes were up-regulated, and the expression of 17 genes was down-regulated in LV-miR-1284 group. One hundred and thirty-eight target genes of miR-1284 were predicted by the bioinformatics website. Compared with invasive cell number of LV-NC-GFP group (168.67±4.55) and control group (170.33±3.08), the ability of invasion ofcells was weakened in LV-miR-1284 group (70.00±2.37). Compared with the liver metastasis rate of LV-NC-GFP group (85.71%) and control group (85.71%), the ability of metastasis of cells was weakened in LV-miR-1284 group (14.29%). Conclusion:The ability of invasion and metastasis of SGC-7901 cells is suppressed by over-expression of miR-1284. The mechanism may be related to regulating the expression ofSUMO1 andJUNgenes.

Effects of propofol on apoptosis in hippocampal neurons of fetal rats in vitro / 中华麻醉学杂志

Objective To evaluate the effects of propofol on apoptosis in the hippocampal neurons of fetal rats in vitro.Methods The isolated hippocampal neurons were seeded into 96-well plates or 24-well plates at a density of 5 × 104 cells/ml.The cells were randomly divided into 5 groups (n =18 each) using a random number table:control group (group C),in tralipid group (group Ⅰ) and propofol 1,10,100 μmol/L groups (P1-3 groups).In group Ⅰ,10％ intralipid was added to the culture media until the final concentration reached 100 μmol/L.In groups P1-3,propofol was added to the culture media until the final concentration reached 1,10 and 100 μmol/L,respectively,and the cells were then incubated for 3 h.The cell apoptosis was assessed by flow cytometry.The expression of Bcl-2 mRNA and caspase-3 mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).The expression of Bcl-2 and actived-caspase-3 protein was determined by Western blot analysis.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,the expression of Bcl-2 mRNA and protein was down-regulated,and the expression of caspase-3 mRNA and actived-caspase-3 protein was up-regulated in P1-3 groups (P ＜ 0.05).There was no significant difference in the parameters mentioned above between group Ⅰ and group C (P ＞ 0.05).Conclusion Propofol induces apoptosis in isolated hippocampal neurons by inhibiting Bcl-2 expression and enhancing caspase-3 activity in fetal rats.

Effect of over-expression of transcription factor CDX2 on proliferation and cell cycle of human gastric cancer cell line SGC-7901 / 中国病理生理杂志

AIM:To study the effect and the molecular mechanism of CDX 2 over-expression on the prolifera-tion, growth and cell cycle of human gastric cancer cell line SGC-7901.METHODS:The SGC-7901 cells in LV-CDX2-GFP group were transfected with the recombinant lentivirus vector LV-CDX2-GFP, the cells in LV-GFP group were trans-fected with the negative control lentiviral vector for the negative control , and the cells in blank control group were without any treatment.The cell proliferation was detected by CCK-8 assay.The cell cycle distribution was analyzed by flow cytome-try.The expression of CDX2, Bax, Bcl-2, cyclin D1 and survivin was determined by semi-quantitative RT-PCR and Wes-tern blotting .RESULTS:Compared with LV-GFP group and blank control group , the proliferation activity of the SGC-7901 cells was significantly lower (P0.05) between LV-GFP group and blank control group .CONCLUSION:Over-expression of CDX2 mediated by lentivirus inhibits the proliferation and growth of human gastric cancer SGC-7901 cells and arrestes the cell cycle at G 0/G1 phase, which may be related to down-regulation of Bcl-2, cyclin D1 and survivin and up-regulation of Bax .

Determination of Dexamethasone in Verdihong Paints by HPLC / 中国药师

Objective:To establish a method for the determination of dexamethasone in Verdihong paints to ensure safety and ef-fectiveness of the clinical medicines. Methods:An HPLC method with a Dikma C18 column (150 mm × 4. 6 mm, 5 μm) was used, the mobile phase was a mixture of methanol-water-glacial acetic acid (80∶20∶0. 5), the detection wavelength was at 240nm, the injec-tion volume was 20 μl, the column temperature was 30℃ and the flow rate was 1. 0 ml·min-1 . Results:The standard curve of dexa-methasone was linear over the range of 3. 0-96. 0μg·ml-1(r=0. 999 7). The average recovery was 99. 45% with RSD of 1. 01%(n=9). Conclusion:The method is simple with good reproducibility, and can be used in the determination of dexamethasone in Verdihong paints.

Reversion of multidrug resistance of human gastric cancer SGC7901/DDP cells by E2F-1 gene silencing / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of E2F-1 gene silencing on multidrug resistance of human gastric cancer SGC7901/DDP cells and its possible mechanisms.</p><p><b>METHODS</b>Gastric cancer SGC7901/DDP cells were seeded in 6 well plates and divided into three groups: the experimental group, blank control and the negative control groups. For the experimental group, the SGC7901/DDP cells were transfected with recombinant lentivirus vector (Lv-shRNA-E2F-1), while the negative control with an control lentiviral vector (Lv-shRNA-NC) and the blank control with no treatment. The E2F-1 protein level was analyzed by Western blot. MTT assay was used to detect the half maximal inhibitory concentration (IC50) of three chemotherapy drugs including adriamycin, 5-fluorouracil (5-Fu) and cisplatine (DDP) of the three cell groups. Flow cytometry (FCM) was used to detect the pump-out rate of adriamycin and apoptosis rate of the three cell groups. Semi-quantitative RT-PCR and Western blot were also used to detect the protein and mRNA levels of multidrug resistance-associated genes (MDR1, MRP) and apoptosis-related genes (c-Myc, Skp2, cyclinD1).</p><p><b>RESULTS</b>The expression of E2F-1 protein in the experimental group was significantly lower than that in the negative control and blank control groups (0.794 ± 0.033 vs. 1.487 ± 0.082 vs. 1.511 ± 0.084, P < 0.01). The IC50 of the three chemotherapy drugs (adriamycin, 5-Fu and cisplatine) in the experimental group was significantly lower than that of the negative control and blank control groups, respectively (P < 0.01). Compared with the negative control and blank control groups, the pump-out rate of adriamycin of the experimental group was significantly declined [(0.16 ± 0.01)% vs. (0.37 ± 0.01)% vs. (0.35 ± 0.02)%, P < 0.01]. However, the apoptosis rate of the experimental group was significantly higher than that of the negative control and blank control groups [(33.82 ± 1.26)% vs. (17.34 ± 0.81)% vs. (13.16 ± 1.06)%, P < 0.01]. The results of RT-PCR and Western blot assays showed that mRNA and protein expressions of five genes (MDR1, MRP, CyclinD1, c-Myc, Skp2) in the experimental group were significantly lower than that in the negative control and blank control groups, respectively (P < 0.01).</p><p><b>CONCLUSIONS</b>E2F-1 gene silencing enhances the chemosensitivity of gastric cancer SGC7901/DDP cells to the chemotherapeutic drugs, directly or indirectly downregulated the expression of MDR1 and MRP, and finally reverses the multidrug resistance of the gastric cancer cells. The mechanism may be associated with the suppression of cyclinD1, c-Myc and Skp2.</p>

Reversion of multi-drug resistance of gastric carcinoma cisplantin-resistant cell SCG7901/DDP by silencing caudal type homeobox 2 / 中华消化外科杂志

Objective To investigate the effects of caudal type homeobox 2 (Cdx2) silence on reservion of multi-drug resistance of gastric carcinoma cisplantin-resistant cell SGC7901/DDP.Methods Gastric carcinoma cisplantin-resistant cells SCG7901/DDP in the logarithmic phase were cultured in the plate,and were divided into the experimental group [gastric carcinoma cells of SGC7901/DDP were infected with a silent Cdx2-recombinanted lentiviral vector (pLL-Cdx2-shRNA)],the negative control group (gastric carcinoma cells of SGC7901/DDP were infected with empty lentiviral vector) and the blank control group (gastric carcinoma cells of SGC7901/DDP were not treated).The protein and mRNA expressions of Cdx2 and apoptosis related genes like c-myc,cyclin D1 and survivin were detected by the Western blot and reverse-transcription PCR,respectively.The sensitivity of the cells in the 3 groups to adriamycin,5-fluorouracil and cisplatium were assessed by MTT.The pump-out rate of adriamycin,cell cycle distribution and apoptosis of the 3 groups were analyzed using flow cytometry.All measurement data were expressed with mean ± standard deviation.Comparison among multi-groups was done by one-way analysis of variance,and comparison between 2 groups was done by SNK-q test.The enumeration data were analyzed using the chi-square test.Results The relative protein expression levels of Cdx2,c-myc,cyclin D1 and survivin were 0.187 ± 0.060,0.086 ± 0.004,0.016 ± 0.005 and 0.276 ± 0.012 in the experimental group,0.535 ± 0.033,0.379 ± 0.006,0.141 ± 0.003 and 0.672 ± 0.009 in the negative control group,and 0.567 ± 0.014,0.354 ± 0.004,0.162 ± 0.008 and 0.517 ± 0.313 in the blank control group,respectively.The relative protein expression levels of Cdx2,c-myc,cyclin D1 and survivin in the experimental group were significantly lower than those in the negative control group and the blank control group (F =247.385,3.353,597.882,98.628,P ＜0.05).The relative mRNA expression levels of Cdx2,c-myc,cyclin D1 and survivin were 0.184 ± 0.010,0.212 ± 0.022,0.045 ± 0.009 and 0.401 ± 0.027 in the experimental group,0.894 ± 0.056,0.538 ± 0.021,0.163 ±0.009 and 0.824 ± 0.016 in the negative control group,and 0.837 ±0.049,0.545 ±0.032,0.157 ±0.010 and 0.782 ±0.056 in the blank control group,respectively.The relative mRNA expression levels of Cdx2,c-myc,cyclin D1 and survivin in the experimental group were significantly lower than those in the negative control group and the blank control group (F =243.776,161.793,138.523,118.426,P ＜ 0.05).The IC50 values detected by MTT of adriamycin,5-flurouracile and cisplatin to gastroc carcinoma cisplantin-resistant cell SCG7901/DDP were (0.12 ± 0.05) mg/L,(0.52 ± 0.13) mg/L and (0.82 ± 0.13) mg/L in the experimental group,(0.33 ± 0.08) mg/L,(4.10.± 1.25) mg/L and (2.81 ± 0.50) mg/L in the negative control group,(0.39 ±0.15)mg/L,(4.05 ± 1.44) mg/L and (3.28 ± 1.03) rng/L in the blank control group,respectively.The pump-out rates of adriamycin of the experimental group,negative control group,and the blank control group were0.21％,0.37％ and 0.35％.Compared with the negative control group and the blank control group,the IC50values of adriamycin,5-fluorouracil and cisplatin in the experimental group were significantly increased,and thepump-out rate of adriamycin was significantly decreased (F =8.101,13.854,15.159,x2 =7.106,P ＜ 0.05).The ratios of cells in the G0/G1 phase were 17.87％,34.71％ and 37.20％ in the experimental group,negative control group and the blank control group,respectively.Compared with the negative control group and blank control group,the ratio of cells in the G0/Gt was significantly decreased (x2=1.055,P ＜ 0.05).The ratio of cells in the G2/M phase in the experimental group was 11.93％,and the apoptosis rate was 31.13％,which were significantly higher than the negative group (0.26％,16.58％) and the blank control group (0.35％,13.18％) (x2=2.249,11.030,P ＜ 0.05).Conclusions Silent Cdx2 can effectively enhance the sensitivity of the SGC7901/DDP cells and the intracellular accumulation concentration of the drugs.Silent Cdx2 can also reverse the multidrug resistance of the SGC7901/DDP cells.

Effects of propofol on intracellular calcium ion concentration and NF-κB activity in hippocampal neurons of fetal rats in vitro / 中华麻醉学杂志

Objective To evaluate the effects of propofol on the intracellular calcium ion concentration ([Ca2+ ]i) and nuclear factor kappa B (NF-κB) activity in hippocampal neurons of fetal rats in vitro .Methods Ten pregnant Sprague-Dawley rats at 16-18 days of gestation ,were sacrificed and the fetal rats were taken out from the abdominal cavity .The hippocampal neurons of the fetal rats were isolated and seeded in culture plates .After being cultured for 9 days ,the neurons were divided into 7 groups ( n=12 each ) using a random number table :control group (C group) ,intralipid group (I group) and propofol 0.1 ,1 ,10 ,100 ,1 000 μmol/L groups (P1-5 groups) .In group I ,10% intralipid was added to the culture media until the final concentration reached 100μmol/L .In P1-5 groups ,propofol was added to the culture media until the final concentration reached 0.1 ,1 ,10 , 100 and 1 000μmol/L ,respectively .The cells were then incubated for 3 h .The [Ca2+ ]i and cellular morphology of hippocampal neurons were examined by laser scanning confocal microscopy before incubation with propofol and within 10 min after the end of incubation with propofol .The expression of NF-κB protein in the nucleus was detected at 7 days after the end of incubation with propofol by Western blot analysis to reflect NF-κB activity . Results Propofol increased [Ca2+ ]i in P2-4 groups ,while decreased [Ca2+ ]i in group P5 ( P0.05 ) .The structure of hippocampal neurons was normal in C ,I and P1 groups .The branchings of axons and dendrites in hippocampal neurons were significantly decreased in P 2-4 groups , while the structure of hippocampal neurons became fuzzy , the cell membrane was destroyed and the axons and dendrites were not seen in group P5 .Conclusion Propofol can produce neurotoxic effects on hippocampal neurons of fetal rats by changing the [Ca2+ ]i and promoting NF-κB activation in vitro .