Objective To study the effects of propofol on enhancement of persistent sodium currents in isolated rat hippocampal CA1 pyramidal neurons induced by ischemia. Methods Whole-cell patch clamp recordings were made from enzymatically isolated SD rat hippocampal CA1 pyramidal neurons. Ischemia was induced by anoxia and glucose deprivation. Results Both propofol 10 umol.L-1 and 100umol . L-1 significantly inhibited the enhancement of persistent sodium currents induced by ischemia and the effect of propofol 100 umol. L-1 was significantly greater than that of propofol 10umol.L-1 . Propofol 1 umol.L-1 didn't have any significant eflect on the enhanced persistent sodium currents induced by ischemia.Conclusion Propofol can inhibit the enhancement of persistent sodium currents induced by ischemia. It may explain the cerebral protective effect of propofol.

Objective To evaluate the effect of intrathecal IL-10 gene on neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve. Methods Sixty female SD rats weighing 230-250 g were anesthetized with pentobarfaital. Right sciatic nerve was exposed at the midthigh level and 4 ligatures were placed on the sciatic nerve with 4.0 catgut at 1mm interval. Intrathecal catheter (PE-10 tubing) were inserted at L5,6 interspace and correct placement was confirmed by outflow of CSF. The animals were randomized into 5 groups ( n = 12): group Ⅰ sham operation; group Ⅱ CCI; group Ⅲ,Ⅳ and Ⅴ received intrathecal (IT) normal saline (NS) (Ⅲ) or pcDNA 3.1 (Ⅳ) or pcDNA 3.1-IL-10 (Ⅴ) 3 days after CCI when the animals developed thermal hyperalgesia. Threshold to noxious thermal stimuli was measured before CCI (baseline), before and 2, 4, 7, 10 and 14 days after IT injection. CSF was collected on 1, 3, 7 and 10 days after IT injection for determination of CSF IL-10 concentration. Six animals were killed on 3rd and 14th days after IT injection respectively in each group and the sciatic nerve, lumbar segment of spinal cord ( L3-6) and hippocampus were isolated and blood was collected for determination of IL-10 mRNA expression (by RT-PCR) (on 3rd day after IT) and TNF-? content (on 14th day after IT) .Results Paw removal latencies were significantly longer 2-14 days after IT injection in group Ⅴ(CCI + pcDNA 3.1-IL-10) than in group Ⅲ (CCI + NS) and Ⅳ (CCI + pcDNA 3.1). The IL-10 mRNA expression in sciatic nerve, lumbar segment of spinal cord and hippocampus was significantly higher 3 days after IT injection while their TNF-? contents were significantly lower 14 days after IT injection in group Ⅴ than in the other 4 groups. The CNS IL-10 concentration on day 1-7 after IT injection was significantly higher in group Ⅴ than in the other 4 groups. Conclusion Intrathecal IL-10 gene injection can ease pain induced by CCI of the sciatic nerve through inhibition of inflammatory response.

Objective:To observe the up-regulation of nuclear Clusterin (nCLU)gene on the biological behaviors of human non-small cell lung cancer cell line A549 .Methods:Sense eukaryotic expression vector of nCLU was constructed by cloning the cDNA of nCLU into pIREShyg3 vector. A549 cells were transfected with pIRES-nCLU and pIREShyg3 vectors by lipofectAMINE~(TM) 2000 mediation, respectively. Stable transfected cells were selected by hygromycin B screening. CCK-8 assay was performed to evaluate the effect of nCLU over-expression on cell proliferation in vitro. The expression level of nGLU protein was examined by Western blotting. Cell cycle distribution was detected by FCM with PI staining. The alteration of migration and metastasis potential of A549 cells before and after nCLU gene transfection were assayed by cell chemotactic migration and invasion test. Results:The proliferation speed of the transfected A549 cell clones stably over-expressing nCLU was slowed down. FCM analyses revealed that the percentage of cells in G_0/G_1 phase dramatically increased from (33.54±2.10)% to (63.31±4.30)%. The cell chemotactic migration and invasion potentials were markedly reduced after nCLU gene transfection (P<0.05). Conclusion:Up-regulation of nCLU can greatly inhibited the proliferation and decreased the migration and invasion capabilities of A549 cells.

ObjectiveTo investigate the role of signal transduction of TLRs in the Henoch-Schonlein purpura (HSP). Methods Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the levels of TLRs(1～10), MyD88, TRAF6, TRIF, IFN-α, IFN-β, IL-6, IL-1β, TNF-α, IP-10, RANTES,iNOS, Blys/April mRNA expression in peripheral blood mononuclear cells and their expression levels were compared using t test., while the concentration of plasma cytokines such as Blys、IFN-α、IFN-β、IL-6、IL-1、TNF-α was measured by enzyme-linked immunosorbent assay(ELISA).Expression levels of those genes were compared using t test. Results①Compared with the control group, the expression levels of TLR1, TLR2,TLR6, TLR5, TLR3, TLR7, TLR9 mRNA were up-regulated significantly(P＜0.01), while no difference of TLR4 was detected (P＞0.05).②Transcription levels of MyDg8(2.47±1.06) vs(0.73±0.22), TRAF6 (2.54±0.72)×10-3vs(0.70±0.20)×10-3, TRIF(3.18±0.86)×10-3vs(0.93±0.35)×10-3 were significantly up-regulated in acute phase of HSP (P＜0.01).③The levels of IFN-α and IFN-β protein and mRNA were remarkable increased (P＜0.01).④ The expression of cytokine/chemotactic factor such as IL-6, IL-1β, IP-10, RANTES,iNOS was higher than that of the control group(p＜0.01), while TNF-αdid not change in children with HSP (P＞0.05). ⑤ It was detected that the expression of Blys/April was higher than that of the control group(P＜0.01). ConclusionExpressions of TLR1, TLR2, TLR6, TIR5, TLR3, TLR7, TLR9, MyD88, TRAF6, and TRIF are up-regulated during acute phase of HSP, suggesting that aberrant activation of TLRs triggered by microbes may be one of the initiating factors of immune aberrance in HSP. Over expression of cytokine/chemotactic factor or Blys/April owning to the aberrant activation of TLRs, may be correlated with immunological pathogenesis of HSP.

Objective To investigate the role of IL-6/STAT3 signaling in Th17/Tr imbalance of Kawasaki disease(KD). Methods Forty-eight children with KD and eighteen age-matched healthy children were consented to participate in this study. Protein concentration of IL-6 in plasma was measured by ELISA. Transcriptional levels of IL-17A, IL-17F, RORγt, Foxp3, SOCS1 and SOCS3 were assessed by real-time PCR. The proportion of CD4+CD25+Foxp3+ regulatory T(Tr) cells and mean fluorescence intensity(MFI) for phosphorylated-STAT3(pSTAT3) protein in CD4+ T cells was analyzed by flow cytometry. A quantitative methylation specific PCR based on SYBR Green was used to evaluate methylation status of CpG islands in SOCS1 exon2, three potential bind sites for STAT3 in 5'-untraslated region(5'-UTR) of SOCS3 in CD4+ T cells. Results (1)Compared with healthy volunteers, plasma IL-6 concentration and MFI for pSTAT3 in CD4+ T cells were elevated significantly during acute phase of KD[IL-6:(54.02±20.58) pg/ml vs (8.72±2.06) pg/ml, P<0.05;pSTAT3 MFI:(55.41±15.08) vs (9.35±3.76), P<0.05], and the two items in KD patients with coronary artery lesion (KD-CAL+) were found to be higher than those in KD patients without coronary artery lesion (KD-CAL-)[IL-6:(84.76±29.35) pg/ml vs (38.65±13.76) pg/ml, P<0.05;pSTAT3 MFI:(72.36±16.81) vs (46.93±13.57), P<0.05]. (2)Transcription levels of IL-17A, IL-17F and RORγt in patients with KD were significantly elevated (P<0.05) while the proportion of CD4+CD25+Foxp3+ Treg and expression levels of Foxp3 were detected to be lower than those in normal controls (P<0.05). The mRNA levels of IL-17A, IL-17F and RORγt in KD-CAL+ group were higher than those in KD-CAL- group(P<0.05), as well as expression level of Foxp3 were found to be lower in KD-CAL+ group(P<0.05). (3)The mRNA levels of SOCS1 and SOCS3 in CD4+ T cells increased significantly during acute phase of KD(P<0.05), while the two items in KD-CAL+ group were lower than those in KD-CAL- group(P<0.05). Furthermore, CpG islands in SOCS1 exon2 and the third potential bind site for STAT3 in SOCS3 5'-UTR were hypomethylated in acute KD, while those in healthy controls were fully demethylated(P<0.05). Demethylation levels of SOCS1 exon2 and the third potential bind site for STAT3 in SOCS3 5'-UTR in KD-CAL+ group were lower than those in KD-CAL- group(P<0.05). CpG islands in the other two bind sites for STAT3 in SOCS3 5'-UTR were fully demethylated among all the groups(P>0.05). ConclusionAberrant activation of IL-6/STAT3 signaling caused by hypomethylation of SOCS1 and SOCS3 might be one contributing factor to unbalance of Th17/Tr in KD.

Objective To investigate the methylation status of Foxp3 gene and its roles in immunological pathogenesis of Kawasaki disease(KD). Methods Thirty children with KD and eighteen agematched healthy children consented to participate in this study. Quantitative methylation specific polymerase chain reaction(MSQP) was used to assess the methylation status of Foxp3 promoter and regulatory T cells specific demethylated region(TSDR) in CD4+ T cells. The proportion of CD4+ CD25 + Foxp3 + regulatory T (Tr) cells was analyzed by flow cytometry. Transcriptional levels of CD4+ CD25 + Foxp3 + Tr associating genes (Foxp3, CTLA4, GITR, LAG3 and CCR8 ) and Foxp3-dependent molecules (UBD and LGAIS3)were measured by real-time PCR. Results ( 1 ) Demethylation level of Foxp3 promoter in CD4 + T cells from patients with KD was lower significantly than that of health subjects( P ＜ 0.01 ), and increased significantly after treated with intravenous gamma globulin therapy(IVIG) (P ＜ 0.01 ). No difference of demethylation level of TSDR region was observed among all groups ( P ＞ 0. 05 ). ( 2 ) The proportion of CD4 + CD25 + Foxp3 + Tr in peripheral blood from patients with KD , as well as mRNA levels of Foxp3 gene in CD4+ T cells, was significantly lower than those of health subjects ( P ＜0. 01 ), and increases significantly after IVIG therapy (P ＜0.01 ). Significant positive correlations between demethylation level of Foxp3 promoter and the proportion of CD4 + CD25 + Foxp3 + Tr , or expression levels of Foxp3 in CD4 + T cells, were observed during acute phase of KD (CD4+CD25+ Foxp3+ Tr: r=0.76, P＜0. 01; Foxp3: r=0.89, P＜0. 01). (3)Transcription level of CD4+ CD25+ Foxp3+ Tr associating factors, such as CTLA4, GITR, LAG3 and CCR8, was significantly down-regulated in acute phase of KD(P＜0. 01 ), and up-regulated to some extent after treated with IVIG(P ＜0. 01 ). Expression levels of Foxp3-dependent molecules UBD and LGALS3 in CD4 + T cells decreased significantly during acute phase of KD (P ＜ 0.01 ), and basically recovered to the levels of health subjects( P ＜ 0.01 ). Conclusion Decrease of demethylation level of Fopx3 promoter is correlated with immune dysfunction in Kawasaki disease.

Objective To investigate the effect of SOCS1 and SOCS3 hypomethylation on homeostasis of Th1/Th2 in Kawasaki disease(KD). Methods Thirty-six children with KD and sixteen age-matched healthy children consented to participate in this study. Protein concentration of IL-6 in plasma was measured by ELISA. Transcriptional levels of SOCS1, SOCS3, T-bet, IFN-γ, GATA3 and IL-4 were assessed by realtime PCR. The proportion of Th1 and Th2 cells, and mean fluorescence intensity(MFI)for phosphorylated STAT3(pSTAT3)protein in CD4+ T cells was analyzed by flow cytometry. A quantitative methylation specific PCR based on SYBR Green was used to evaluate methylation status of CpG islands in SOCSl exon2, and three potential binding sites for STAT3 in 5'-untraslated region(5'-UTR)of SOCS3 in CD4+T cells. Comparisons between groups were performed with t-test. Results ①Compared with healthy volunteers, plasma IL-6 concentration[(51.8±16.3)pg/ml vs(8.6±2.0)pg/ml, respectively]and MFI for pSTAT3[(52±14)vs(10±4), respectively]in CD4+ T cells were elevated significantly during acute phase of KD(P＜0.05), and the two items in KD patients with coronary artery lesion(KD-CAL+)were found to be higher than those in KD patients without coronary artery lesion(KD-CAL-)[IL-6:(87.2±27.4)pg/ml vs(36.2±12.8)pg/ml, P＜0.05; pSTAT3 MFI:(75±15)vs(42±11), P＜0.05]. ② The proportions of Th1 and Th2 cells and transcription levels of Th-associating factors(T-bet, IFN-γ, GATA3 and IL-4)in CD4+ T cells increased significantly in acute KD(P＜0.05), while the rate of Thl div Th2 in KD patients was found to be lower than that in normal controls(P＜0.05). In addition, the proportions of Th1 and Th2 cells and expressions levels of Th-associating factors in KD-CAL+ group were higher than those in KD-CAL-group, as well as the rate of Thl div Th2 cells in KD -CAL+ group were lower than that in KD-CAL- group(P＜0.05). ③ The mRNA levels of SOCSl and SOCS3 in CD4+ T cells increased significantly during acute phase of KD(P＜0.05), while the two items in KDCAL+ group were lower than those in KD-CAL- group(P＜0.05). Furthermore, CpG islands in SOCSl exon2 and the third potential binding site for STAT3 in SOCS3 5'-UTR were hypomethylated in acute KD, while those in healthy volunteers were fully demethylated(P＜0.05). Demethylation levels of the two items mentioned above in the KD-CAL+ group were lower than those in the KD-CAL-group(P＜0.05). CpG islands in the other two binding sites for STAT3 in SOCS3 5'-UTR were fully demethylated among all the groups(P＞0.05).Conclusion Relative insufficiency of SOCS1 and SOCS3 expression caused by hypomethylation may be one contributing factor for the imbalance of Th1/Th2 in KD.

Aim To explore the concentration range of organic solvent which can both effectively increase solubility of the difficult soluble medicine monomer, and have low toxicity to cells, and to clarify the influence of different concentration of ethanol on curcumol efficacy.Methods Different DMSO and ethanol concentrations were diluted in culture medium and incubated with cells A549, NCI-H460, NCI-H1299, NCI-1650, LTEP-a2 and SPC-A1 for 12 h, 24 h, or 48 h, cell viability was tested by a colorimetric assay with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide(MTT), and different concentrations of ethanol with/without different concentrations of curcumol were also prepared with culture medium, then incubate with A549 and NCI-H460 cells for 24 h, and cell viability was tested by MTT as well.Results Within 48 h the solution with 0.008(V/V) DMSO or less had no significant effect on the cell A549 compared with control group, for NCI-H1650 the concentration was 0.004(V/V) or less, for NCI-H460 the result turned to be 0.002(V/V) or less, and the solution with DMSO below 0.001(V/V) had significant effect on the three other cells, NCI-H1299, LTEP-a2 and SPC-A1.While within 48 h, the liquor with 0.004(V/V) ethanol or less did not exhibit significant cytotoxic effect on the cell A549, for NCI-H460 and NCI-H1650 the result of ethanol concentration became 0.002(V/V) or less, for NCI-H1299 the data was 0.001(V/V) or less, and the liquor with ethanol below 0.001(V/V) showed significant cytotoxic effect on LTEP-a2and SPC-A1.When the proportion of ethanol in solution was below 0.01, it has no cytotoxic on cell A549 and NCI-H460, and the curcumol solution prepared with this kind of ethanol solution only represented the efficacy of curcumol on the cells.Since the solution with 0.01(V/V) ethanol dissolved the curcumol better, the cell viability changed from about 100% to about 35%.Conclusions Different organic solvent expressed different toxicity to the same cell, and the sensitivity of different cells to one organic solvent is dissimilar.and DMSO could be the optimum solvent for A549 and NCI-H1650, while the optimum solvent of NCI-H1299 is ethanol, for NCI-H460 it could be both and DMSO and ethanol, and DMSO and ethanol is not suitable for LETP-a2, SPC-A1 to be solvent.

[Summary] The proportions of CD19+CD24 hi CD38 hi regulatory B cells ( Breg ) and CD4+CD25+Treg in peripheral blood of children with type 1 diabetes mellitus ( T1DM) were detected by flow cytometric analysis. The concentration of interleukin 10 (IL-10) protein was measured by ELISA. The mRNA expression levels of Foxp3, cytotoxic T lymphocyte-associated antigen-4 ( CTLA-4 ) , and glucocorticoid-induced tumor necrosis factor receptor ( GITR) in CD4+T cells and IL-10 in CD19+ B cells were evaluated using real-time PCR. The results showed that the proportions of CD19+CD24hiCD38hiBreg in peripheral blood, the mRNA and protein levels of IL-10 in B cells from patients with T1DM were lower than those from healthy controls (P<0. 05). The mRNA expression levels of Treg associated factors such as Foxp3, CTLA-4, and GITR were lower in CD4+ T cells from children with T1DM compared with the controls(P<0. 05). These results suggest that Breg cell deficiency and dysfunction might be one of the important factors causing cellular immune dysfunction in patients with T1DM.

Objective To evaluate the role of mitochondrial fission in anoxia-reoxygenation injury to rat hippocampal neurons.Methods Neurons were enzymatically isolated from hippocampi of newborn Sprague-Dawley rats (less than 24 h old).The primary hippocampal neurons were cultured and seeded in 25 mm × 25 mm culture flasks at a density of 7 × 105/ml.The cultured neurons were randomly assigned into 3 groups (n =18 each) using a random number table:control group (C group),anoxia-reoxygenation group (I/R group),and mitochondrial fission inhibitor mdivi-1 group (M group).In group I/R,the vehicle dimethyl sulfoxide (DMSO,final concentration ＜ 0.1％) was added prior to anoxia and the cells were then incubated for 40 min.In group M,mdivi-1 (dissolved in DMSO,final concentration of DMSO ＜ 0.1％) was added prior to anoxia and the cells were then incubated for 40 min.The hippocampal neurons were subjected to oxygen-glucose deprivation (OGD) for 6 h followed by restoration of O2 supply for 20 h.After 20 h of reoxygenation,the level of reactive oxygen species (ROS) (by ELISA),cell apoptosis (using flow cytometry),and expression of mitochondrial fission protein Drp1,Bcl-2 and Bax (by Western blot) were measured.The apoptosis rate and the ratio of Bcl-2 to Bax were calculated.Results Compared with C group,ROS content and apoptosis rate were significantly increased,the expression of Drp1 and Bax was up-regulated,the expression of Bcl-2 was down-regulated,and the ratio of Bcl-2 to Bax was decreased in I/R group (P ＜ 0.05).Compared with I/R group,ROS content and apoptosis rate were significantly decreased,the expression of Drp1 and Bax was down-regulated,the expression of Bcl-2 was up-regulated,and the ratio of Bcl-2 to Bax was increased in M group (P ＜ 0.05).Conclusion Mitochondrial fission is involved in anoxia-reoxygenation injury to rat hippocampal neurons via mitochondria-mediated apoptotic pathway.