Aim To investigate the role of T type calcium channel of spinal cord and supraspinal on the pain threthold of the rats following chronic constriction injury(CCI) of the sciatic nerve.Methods Intrathecal and lateral ventricle injection were employed in this study.With Von Frey hair and radiant thermal stimulator,we measured the mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL)of the rats after injected mibefradil.Results The rats of CCI group formed steady mechanical and heat hyperaglsia from the third day after operation to the end of this study.Administered intrathecally mibefradil 50,100,200 ?g can increase the CCI rats MWT and TWL.However,mibefradil administered lateral ventricle can reduce the CCI rats MWT and TWL.Conclusion Blocking T type calcium channel of spinal can inhibit mechanical and heat hyperalgesia of the CCI rats,However,bloking the T type calcium channel of supraspinal can enhance the mechanical and heat hyperalgesia of the CCI rats.

ObjectiveTo investigate the role of T-type calcium channel in the spinal neurotoxicity of intrathecal (IT) lidocaine in rats.MethodsForty-eight adult male SD rats in which IT catheter was successfully implanted,weighing 230-270 g,were randomly divided into 4 groups ( n =12 each):dimethyl sulfoxide (DMSO)group (group D),lidocaine group (group L),mibefradil + lidocaine group (group M),normal saline + lidocaine group (group N).Another 12 rats served as control group (group C).DMSO and 10％ lidocaine 20μl were injected intrathecally in groups D and L respectively.After mibefradil 200 μg/10μl and normal saline 10 μl were injected intrathecally in groups M and N respectively,10％ lidocaine 20 μl was injected intrathecally in the two groups.The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured before IT injection and at 2,4,8 and 12 h and 1,2,3,4 and 5 d after IT injection (T0-9).Four rats were sacrificed at T6 in each group and their lumbar enlargements were removed for microscopic examination.ResultsCompared with group C,no significant change in MWT and TWL was found at each time point in group D,MWT was significantly increased at T1-8 and TWL was significantly prolonged at T1-7 in groups L and N,and MWT was significantly increased at T1-6 and TWL was significantly prolonged at T1-6 in group M ( P ＜ 0.05 ).Compared with groups L and N,MWT was significantly decreased at T1-4 and TWL was significantly shortened at T1-4 in group M ( P ＜ 0.05).Pathological injury was significantly reduced in group M as compared with groups L and N.ConclusionT-type calcium channel is involved in the spinal neurotoxicity of IT lidocaine in rats.

ObjectiveTo investigate the effects of sevoflurane on inhibition of invasive activity and migration of human adenocarcinoma cell line A549 by cisplatin.MethodsThe.human lung adenocarcinoma cell line A549 was obtained from Shanghai Cell Biology Institute,Chinese Academy of Sciences and cultured in RPMI 1640 culture medium containing 10％ fetal calf serum.The cells were inoculated in culture plate and cultured for 24 h and randomly divided into 4 groups:control group; 2.5 ％ sevoflurane group ; cisplatin group and cisplatin + 2.5 ％sevoflurane group.In groups sevoflurane,cisplatin and cisplatin + sevoflurane the cells were exposed to 2.5％sevoflurane or/and cisplatin 10μmol/L for 4 h respectively.The invasive activity of the cells was evaluated by Transwell chamber assay.The migration of the cells was determined by wound healing assay.The expression of MMP-2,MMP-9,Ezrin,and Fascin in the cells was detected by Western blot.ResultsBoth 2.5％ sevoflurane and cisplatin depressed invasive activity and migration of the A549 cells and down-regulated MMP-2,MMP-9,Ezrin and Fascin expression in A549 cells.The inhibitory effects of cisplatin on the A549 cells were potentiated by 2.5 ％ sevoflurane.ConclusionSevoflurane can enhance the inhibition of invasive activity and migration of human adenocarcinoma cell line A549 by cisplatin.

Objective To investigate the effects of different concentrations of sevoflurane on Survivin expression in human adenocarcinoma cell line A549. Methods A549 cells were obtained from Shanghai Cell Biology Medical Research Institute, Chinese Academy of Sciences and inoculated in 96 well culture plate. After being cultured for 24 h, the cells were randomly divided into 4 groups: Ⅰ , Ⅱ , Ⅲ and Ⅳ groups exposed to 95 % O2 -5 %CO2,1.7%, 3.4% and 5.1% sevoflurane respectively. A549 cells were exposed to sevoflurane for 2, 4 and 6 h respectively and then cultured for another 48 h in Ⅱ , Ⅲ and Ⅳ groups. Proliferation of A549 cells were measured by methyl thiazolyl tetrazolium (MTT) assay, and apoptosis was detected with flow cytometer at 48 h after 2, 4 and 6 h sevoflurane exposure. The expression of Survivin in A549 cells was determined by Western blot analysis at 48h after 4 h sevoflurane exposure. Results The rate of proliferation inhibition and percentage of apoptotic cells were significantly higher while the expression of Survivin was significantly lower in a concentration-dependent manner in Ⅱ , Ⅲ and Ⅳ groups as compared with group Ⅰ . Conclusion Sevoflurane can inhibit proliferation and induce apoptosis of A549 cells by inhibition of Survivin expression.

Objective To investigate the effects of isoflurane and sevonumne on apoptosis and expression of CD44 and CD54 in human lung cancer cell line A549.Methods Human lung cancer A549 cells were obtained from Shanghai Cell Biology Medical Research Institute,Chinese Academy of Sciences,and inoculated in 24 well culture plate.After being cultured for 24 h the cells were randomly divided into 3 groups:group Ⅰ control(group C);group Ⅱ isoflurane (group Iso) and group Ⅲ sevoflurane (group Sev).A 549 cells were exposed to 1.7% isoflurane and 2.5%sevoflurane for 4 h respectively in group Iso and Sev respectively,and were then cultured for another 24 h.Apoptosis and expression of CD44 and CD54 in A549 cells were detected with flow cytometer at 0 (T0),2 h(T1) and 4 h(T2) of and 24 h after(T3) exposure to isoflurane and sevoflurane.Results The percentage of apoptotic cells wag significantly higher at T2 and T3 in group Iso than in group C.The percentage of apoptotic cells was significantly higher at T1,T2 and T3 in group Sev than in group Iso and C.The expression of CD44 and CD54 at T1,T2 and T3 was significantly decreased as compared with the baseline at T0 in group Iso and Sev and was significantly lower in group Iso and Sev than in group C.Conclusion Isoflurane and sevoflurane can induce apoptesis of human lung cancer cell line A549, and sevoflurane is more effective. Isoflurane and sevoflurane can inhibit the expression of CD44 and CD54 of human lung cancer cell line A549.

Objective To evaluate the effects of sevoflurane on invasion and migration of mouse lung cancer cells induced by hypoxia.Methods Mouse Lewis lung cancer cells were inoculated in the culture plate.After being cultured for 24 h,the cells were randomly divided into 3 groups (n=18 each) using a random number table:control group (group C),hypoxia group (group H) and hypoxia+ 2％ sevoflurane group (group HS).Cells were exposed to 95％ air-5％CO2 (2 L/min) for 4 h in group C.Cells were exposed to 94％ N2-5％CO2-1％ O2 for 4 h in group H.In group HS,cells were exposed to 2％ sevoflurane and 94％ N2 (2 L/min) for 4 h.The invasion of cells was determined by Transwell assay,and the invaded cells were counted.The migration of cells was evaluated by wound healing assay,and cell migration rates were calculated.The expression of Beclin 1 and LC3 Ⅱ protein in cells was detected by Western blot.Results Compared with group C,the number of invaded cells and cell migration rates were significantly increased,and the expression of Beclin Ⅰ and LC3 Ⅱ was up-regulated in H and HS groups.Compared with group H,the number of invaded cells and cell migration rates were significantly decreased,and the expression of Beclin 1 and LC3 Ⅱ was down-regulated in group HS.Conclusion Sevoflurane can inhibit the invasion and migration of mouse lung cancer cells induced by hypoxia,and inhibition of autophagy is involved in the mechanism.

Objective To evaluate the effects of sevoflurane on the expression of heparanase ( HPA) and fascin in lung carcinoma cells of mice. Methods Mouse LLC cells were inoculated in the culture plate. After being cultured for 24 h, the cells were equally and randomly divided into 4 groups using a random number table: control group ( group CC) , 1% sevoflurane group ( group Sev1 ) , 2% sevoflurane group ( group Sev2 ) , and 3% sevoflurane group ( group Sev3 ) . Cells in Sev1-3 groups were exposed to 1%, 2% and 3% sevoflurane, respectively, for 4 h, while cells in group CC were not exposed to sevoflurane, and all the cells were then cultured for another 24 h in an incubator. The invasion of cells was determined by Transwell invasion assay, and the invaded cells were counted. The migration of cells was determined by wound healing assay, and cell migration rates were calculated. The expression of HPA and fascin in cells was detected by Western blot. Results Compared with group CC, the number of invaded cells and cell migration rates were gradually decreased, and the expression of HPA and fascin was gradually down?regulated with increasing concentrations of sevoflurane in Sev1-3 groups. Conclusion The mechanism through which sevoflurane inhibits the metastasis of mouse lung carcinoma cells is associated with down?regulated expression of HPA and fascin.

Objective To evaluate the role of C-Jun N-terminal kinase (JNK) signal transduction pathway in spinal neurotoxicity induced by lidocaine in rats.Methods Seventy-two adult male Sprague-Dawley rats,weighing 220-260 g,were randomly divided into 6 groups (n =12 each):control group (group Ⅰ),sham operation group (group Ⅱ),JNK inhibitor group (group Ⅲ),dimethyl sulfoxide (DMSO) group (group Ⅳ),lidocaine group (group Ⅴ),and JNK inhibitor and lidocaine group (group Ⅵ).Group Ⅰ received no treatment.Intrathecal catheter was placed in the subarachnoid space in group Ⅱ.SP600125 25 μg and DMSO 20 μl were injected intrathecally in Ⅲ and Ⅳ groups,respectively.In group Ⅴ,10％ lidocaine 20 μl was intrathecally injected.SP600125 25 μg was injected intrathecally and 30 min later 10％ lidocaine 20 μl was injected intrathecally in group Ⅵ.Paw withdrawal threshold to yon Frey filament stimulation (PWT) and paw withdrawal latency to nociceptive thermal stimulation (PWL) were measured before intrathecal catheter was implanted (T0),before intrathecal administration (T1) and at 4,8 and 12 h and on 1,2,3,4,5 and 6 days after intrathecal administration (T2-10).At 24 h after intrathecal administration,4 rats were randomly chosen from each group and sacrificed.Their lumbar enlargements were removed for determination of phosphorylated JNK (p-JNK) expression (using Western blot) and neuronal apoptosis (by TUNEL).The apoptotic index was calculated.Results Compared with group Ⅰ,no significant difference was found in MWT and TWL in Ⅱ,Ⅲ groups and expression of p-JNK in Ⅱ and Ⅳ groups (P ＞ 0.05),MWT at T2-4,6-8 and TWL at T2-4,7 in group Ⅴ and MWT at T2-6 and TWL at T2-5 in group Ⅵ were significantly increased,the expression of p-JNK was down-regulated and the apoptotic index was decreased in group Ⅲ (P ＜ 0.05),and the expression of p-JNK was up-regulated and the apoptotic index was increased in Ⅴ and Ⅵ groups (P ＜ 0.05).Compared with group Ⅴ,MWT and TWL were significantly decreased,the expression of pJNK was down-regulated and the apoptotic index was decreased in group Ⅵ (P ＜ 0.05).Conclusion Activation of JNK signal transduction pathway is involved in spinal neurotoxicity induced by lidocaine in rats possibly through promoting neuronal apoptosis in the spinal cord.

Objective To evaluate the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in the up-regulation of expression of Cav3.2 T-type calcium channels in spinal cord in a rat model of neuropathic pain (NP).Methods Forty-eight male Sprague-Dawley rats,aged 3 months,weighing 220-250 g,were randomly divided into 6 groups (n =8 each)∶ sham operation group (group S),group NP,dimethyl sulfoxide group (group D) and different concentrations of a specific CaMK Ⅱ inhibitor KN93 groups (groups K1-3).NP was produced by chronic compression of dorsal root ganglion.The rats in groups D and K1-3 received a single intrathecal injection of dimethyl sulfoxide and KN93 15,30,60 nmol/L (10 μl),respectively,on 5th day after NP.Paw withdrawal threshold to von Frey filament stimulation (MWT) and paw withdrawal latency to thermal nociceptive stimulus (TWL) were measured before NP,before intrathecal injection on 5th day after NP,and at 30 and 60 min and 3,6 and 8 h after intrathecal injection on 5th day after NP (T1-7).The rats were sacrificed after the measurement of pain threshold at T7 and their lumbar enlargements were removed to detect the expression of Cav3.2 mRNA and protein using Western blot and RT-PCR.Results Compared with group S,MWT was significantly decreased,TWL was shortened and the expression of Cav3.2 mRNA and protein was up-regulated in NP,D and K1-3 groups (P ＜ 0.05).Compared with NP group,MWT was significantly increased,TWL was prolonged and the expression of Cav3.2 mRNA and protein was down-regulated in a concentration-dependent manner in K1-3 groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in group D (P ＞ 0.05).Conclusion CaMK Ⅱ is involved in the development and maintenance of chronic NP by up-regulating the expression of Cav3.2 T-type calcium channels in rat spinal cord.