Effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil induced apoptosis in human colon carcinoma cell lines

AIM: To investigate the effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil (5-FU) induced apoptosis in colon carcinoma cell lines.METHODS: The expression of the αvβ6 integrin in colon carcinoma cell lines HT-29 and WiDr cells was analyzed by flow cytometry. The apoptosis induced by 5-FU and the effects of αvβ6 integrin-mediated cell adhesion on 5-FU induced cell apoptosis were measured by enzyme-linked immunosorbent assay (ELISA) method and acridine orange-ethidium bromide (AO-EB) double fluorescent dye staining.RESULTS: Both the colon carcinoma cell lines HT-29 and WiDr cells expressed the αvβ6 integrin. The percentages of HT-29 and WiDr cells expression were 80.82% and 82.96%. 5-FU induced the apoptosis of colon carcinoma cell lines HT-29 and WiDr. The result of ELISA method displayed that enrichment factor (EF) of HT-29 and WiDr cells planted on fibronectin (FN)-ligand of αvβ6 integrin was lower significantly than the EF of HT-29 and WiDr cells planted on non-integrin ligand polylisin (1.11±0.04 vs 3.68±0.03, 1.09±0.02 vs 3.72±0.02, P<0.01) after cultured in medium containing 20 mg/L 5-FU for 48 h. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again, the EF of HT-29 and WiDr cells was higher significantly than those directly planted on FN without being blocked by αvβ6 integrin antibody (2.12±0.04 vs 1.11±0.04, 2.14±0.03 vs 1.09±0.02, P<0.01). The AO-EB double fluorescent dye staining displayed that the apoptosis percents of HT-29 and WiDr cells planting on FN were (5.6±1.1)% and (5.3±0.7)%, which were lower significantly than those planting on polylisin (37.0±1.4)%, (38.5±0.9)%, P<0.01. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again the percents of HT-29 and WiDr cells apoptosis were (19.5±1.2)% and (20.0±0.7)%, which increased significantly compared with those directly planting on FN without being blocked by αvβ6 integrin antibody (P<0.01). CONCLUSION: Colon carcinoma cell lines HT-29 and WiDr cells expressed αvβ6 integrin. The cell adhesion with FN mediated by αvβ6 integrin inhibits 5-FU-induced colon carcinoma cell apoptosis. The results suggest that cell adhesion may enhance drug resistance in colon carcinoma cell lines through inhibiting the cell apoptosis.     

Involvement of caspase-3 and p38MAPK in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells

AIM: To investigate the function of caspase-3 and mitogen-activated protein kinases (MAPKs) in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells. METHODS: Allogeneic CD8+T cells were isolated from PBMC by positive selection using magnetic beads coated with anti-CD8 antibody. After cocultured with allogeneic CD8+T cells, apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) were detected by AnnexinV-FITC labeling. Western blotting was used to examine the change of MAPK and caspase-3 expression in the vascular endothelial cells. The influence of SB203580 (inhibitor of p38MAPK), SP600125 (inhibitor of JNK), PD98059 (inhibitor of ERK), Z-DEVD-FMK (a caspase-3-specific peptide inhibitor) on apoptosis was also examined. RESULTS: At 24 h and 48 h time-point, the apoptosis rates of HUVECs were 41.7%±10.1% and 29.4%±8.3%, respectively (P<0.01, vs untreated HUVECs); the apoptosis rates of HDMECs were 28.9%±7.2% and 15.2%±4.8%, respectively (P<0.01, vs untreated HDMECs). These effects were largely prevented by Z-DEVD-FMK and SB203580 (P<0.05). Allogeneic CD8+T cells enhanced cleavage of caspase-3 and led to p38MAPK phospholation. CONCLUSION: Caspase-3 and p38MAPK mediate allogeneic CD8+T cells-induced apoptosis of vascular endothelial cells.     

Effects of recombinant human CD40 ligand on biological behavior of ovarian cancer SKOV3 cells in vitro

AIM: To investigate the effect of recombinated human CD40 ligand (rhCD40L) on the biological behavior of ovarian cancer SKOV3 cell line in vitro.METHODS: After the SKOV3 cells were incubated with different concentrations of rhCD40L for various times, the cell proliferation was determined by MTT assay.The expression of the co-stimulatory molecules or adhesion molecules on SKOV3 cells and the changes of tumor necrosis factor receptor associated factor (TRAFs) inside the cells were measured by flow cytometry and direct immunofluorescence.Annexin V and PI dual color label assay were used to detect cell apoptosis or death in culture contained with rhCD40L.RT-PCR assay was employed to determine the change of apoptosis related gene c-myc, bcl-2 and bcl-xl expression in SKOV3 cells.RESULTS: rhCD40L inhibited proliferation of SKOV3 cells at concentration of 100 μg/L (0.65±0.10 vs 0.81±0.05) and reached a peak at concentration of 10 mg/L (0.13±0.12 vs 0.83±0.15, P＜0.01).The inhibitory effects showed a dose dependent manner.Cell cycle analysis showed that cell division was blocked in G1 phase.Increasing proportion of apoptosis of SKOV3 cells was related to up-regulation of CD95 expression (42.4% vs 59.2%, P＜0.05) and down-regulation of anti-apoptosis genes such as bcl-2 and bcl-xl expressions after incubation with rhCD40L.TRAF 2, 5 and 6 expressed highly in SKOV3 cells.The expression of TRAF 2 (81.3%±9.2% vs 50.4%±5.3%，P＜0.05), TRAF5 (47.2%±7.2% vs 7.2%±2.1%, P＜0.01) and TRAF6 (44.5%±6.3% vs 5.1%±1.1%, P＜0.01) was down-regulated and expression of TRAF 3 (25.2%±6.2% vs 68.8%±5.3%, P＜0.01) was up-regulated after co-culture with rhCD40L, but there was no effects found on the expression of TRAF 1 (4.3%±1.2% vs 5.1%±1.4%) and TRAF4 (7.4%±1.2% vs 8.1%±1.4%).CONCLUSION: By down-regulating expression of bcl-2, bcl-xl and changing expression profile of TRAF, rhCD40L inhibits the growth of SKOV3 cells by blocking the cell cycle progress in G1 and promotes the cells to apoptosis.     

Kinetic expressions of PD-L1 and PD-L2 on the surface of human lymphocytes and monocytes

AIM: To investigate the expression kinetics of PD-L1 and PD-L2 on the surface of the resting and activated B/T cells as well as monocytes from healthy human peripheral blood. METHODS: Fluorescent antibody staining together with flow cytometry were used to detect the percentages of the resting as well as the activated B cells and T cells that expressed PD-L1 and PD-L2. Meanwhile the percentages of the resting and activated monocytes that expressed PD-L2 were determined. RESULTS: Both resting B cells and T cells did not express PD-L1 on their surface, however PD-L1 expression was significantly up-regulated on the surface of the activated B cells after 6 h stimulation with LPS or pokeweed mitogen (PWM), and the percentages of B cells that expressed PD-L1 reached a plateau at 24 h, which were (46.26±10.71)% with LPS and (43.67±6.14)% with PWM stimulation, respectively. No markedly change of PD-L1 expression on the surface of the activated T cells after stimulation with LPS was observed, but upregulation of PD-L1 expression was observed when stimulation with PWM. The percentages of T cells that expressed PD-L1 reached a plateau at 24 h, which was (25.42±9.23)%. PD-L2 expression was not found on the resting as well as the activated B cells and T cells. In addition, the resting monocytes did not express PD-L2. Combination of INF-γ plus LPS markedly induced the PD-L2 expression, and the percentages of monocytes that expressed PD-L2 reached a peak at 48 h, which was (28.70±14.22)%. CONCLUSION: The activated lymphocytes only express PD-L1, reaching a plateau at 24 h. PD-L2 is expressed on the surface of the activated monocytes, reaching a peak at 48 h.     

Inhibitory effects of a cyclooxygenase-2 inhibitor,NS398,on cancer cells

AIM:To study the mechanism of growth inhibitory effect of a selective cyclooxygenase-2 (COX-2) inhibitor,NS-398,on cancer cells.METHODS:The esophageal cancer cell line (EC9706),which expresses COX-2 constitutively,and hepatocellular carcinoma cell line (SMMC7721),which expresses no COX-2,were studied.The cell lines were incubated with NS-398 at doses of 10,20,50,100 μmol/L for 24 h,48 h and 72 h.Antiproliferation effect was measured by ［3H］-TdR incorporation.The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis.Survivin was detected by immunocytochemical technique.RESULTS:The growth inhibition was induced by NS398 in a dose- and time-dependent manners in both cell lines.FCM analysis revealed a high sub-G1 cell peak in EC9706 group and agarose electrophoresis showed marked apoptosis ladder pattern.However,no apoptosis was observed in SMMC7721 cells treated with NS-398.The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23±1.08)% and (3.05±0.15)% (P<0.01).After 24 h incubation with NS-398 at concentration of 100 μmol/L,the expression of survivin was markedly reduced in EC9706,no change was observed in SMMC7721.CONCLUSION:NS-398 suppresses cell growth in cancer cell lines by different mechanism.NS-398 suppresses cell growth and increases apoptosis in the cancer cells that expresses COX-2.     

Effect of FAK-related non-kinase on apoptosis in hepatic stellate cells

AIM: To evaluate the inhibitory effect of FRNK on the phosphorylation of FAK and apoptosis in hepatic stellate cells (HSCs). METHODS: After stimulated with fibronectin, HSCs was transfected with FRNK plasmid by cationic liposome method. The apoptosis of FRNK-induced HSCs was examined by Annexin-V/propidium iodide double-labeled flow cytometry (FCM), gel electrophoresis and transmission electron microscope. The protein levels of FRNK, FAK and p-FAK (Tyr397) in HSCs were assayed by Western blotting, and RT-PCR was used to detect the expression of mRNA. RESULTS: The expression of FRNK was enhanced and the phosphorylation of FAK was inhibited after FRNK was transiently transfected into HSCs in vitro. The apoptotic rate in HSCs exposed to FRNK plasmid for 48 h was higher than that in the non-FRNK plasmid group ［(25.37±1.92) % vs (9.28±1.05) %, P<0.01］, and accompanied by a significant increase in caspase-3 activity both in the protein and in the mRNA level ［(264.17±12.60 vs 185.82±9.69), P<0.01; (4.19±0.48 vs 1.07±0.27), P<0.01］. CONCLUSION: In HSCs, the expression of FRNK is enhanced and the phosphorylation of FAK is inhibited after FRNK transfection. FRNK induces the HSCs apoptosis.     

Effect of proteasome inhibitor MG132 on proliferation and cell cycle distribution of NK/T cell lymphoma cells

AIM: To study the influence of MG132 on the proliferation and cell cycle distribution of NK/T cell lymphoma cells, and to investigate the potential role of proteasome inhibitor on the treatment of NK/T cell lymphoma. METHODS: NK/T cell lymphoma cells HANK1 were treated with proteasome inhibitor MG132, and the proliferation was evaluated by MTT assay. The morphological changes were observed under inverse microscope. The cell cycle distribution and apoptosis were detected using flow cytometry. RESULTS: The growth inhibitory rate of HANK1 cells was(57.72±7.44)% after cultured for 24 h with 1 μmol/L MG132 and was just(3.98±0.07)% after cultured for 24 h with 0.1 μmol/L MG132. The positive relationship between the concentration of MG132 and growth inhibitory rate was observed. On the other hand, after cultured for 24 h with 1μmol/L MG132, the cells in G1 and G2 phases were(72.33±3.44)% and(12.86±1.29)%, respectively, much higher than those in control group(63.63%±2.29% and 7.94%±1.91%, respectively). The early and late apoptosis rates in MG132 group were 33.57%±2.10% and 16.66%±0.47%, respectively, much higher than that in control group (7.18%±0.82% and 3.81%±1.06%, respectively). CONCLUSION: MG132 inhibits cell proliferation and induces cell cycle arrested at G1 and G2 phases, and cell apoptosis in NK/T cell lymphoma cells in a concentration dependent manner. Proteasome inhibitor may be a good drug to treat patients with advanced NK/T cell lymphomas.     

Mechanism of 13-methyltetradecanoic acid in inducing bladder cancer T24 cell apoptosis in vitro

AIM: To study the effect of 13-methyltetradecanoic acid (13-MTD) on bladder cancer T24 cell apoptosis and the underlying mechanisms. METHODS: T24 cells were treated with 13-MTD at different concentrations. MTT cell proliferation assay was used to observe the inhibitory effect. The cell cycle and cell apoptosis were displayed by FCM. Apoptosis index was investigated by TUNEL assay. Western blotting was used to detect the expression of apoptosis related proteins. RESULTS: p38 and JNK phosphorylation were presented 2 h after stimulated by 13-MTD, but AKT phosphorylation were inhibited. Cytochrome C was released from the intermembrane space of mitochondria 8 h later. No change of FADD and p-FADD was observed. Several downstream caspase substrates (PARP, lamin B, RB) were cleaved in 12 h. CONCLUSION: In the course of T24 cell apoptosis induced by 13-MTD, JNK/SAPK and p38MAPK signaling pathway are activated and the PI3K/AKT signaling pathway is inhibited. The substrates of caspase: PARP, Rb, lamin B are cleavaged by activated caspase enzymes to promote T24 cell apoptosis.     

Abnormal T-cell receptor signal pathway in murine autoimmue cardiom yophy induced with adenine nucleotide translocase

AIM: To explore the molecular mechanism in the pathogenesis of dilated cardiomyopathy (DCM) by analyzing the expression of T cell signaling molecules in mice with autoimmune DCM. METHODS: Mouse DCM model was induced by immunizing the animals with adenine nucleotide translocase (ANT) synthetic peptides. P56lck in T cells was detected with real-time fluorescent quantitative PCR in both DCM-group and the sham-immunized controls. At the same time, flow cytometry was used for quantity of Th cell intracellular cytokine IFN-γ and IL-4, ELISA for examining the level of serum anti-ANT antibody, immune histochemistry for investigating the expression of CD45 in Th cells. RESULTS: The mRNA expression of P56lck (1 369.51±874.05 vs 47.93±10.21, P<0.01), the percentage of IFN-γ and IL-4 (especially IL-4) (8.27±1.29 vs 5.58±0.59, P<0.01; 9.93±1.53 vs 2.05±0.21, P<0.01), the level of anti-ANT autoantibody (0.105±0.015 vs 0.006±0.002, P<0.01) and expression of CD45 (0.154±0.021 vs 0.026±0.008, P<0.01) were all elevated significantly in DCM-group compared with the controls. CONCLUSION: Impairment of T cell receptor (TCR) signal transduction pathway plays an important role in the development of DCM induced with ANT synthetic peptides in mice.     

Sodium valproate induces cycle arrest,apoptosis and elevates p21WAF1 mRNA expression in chronic myeloid leukemia cell line K562

AIM:To investigate the effects of sodium valproate (VPA) on the cell cycle and apoptosis of chronic myeloid leukemia cell line K562, and to explore the possible mechanisms. METHODS:K562 cells were treated with VPA. Cell cycle and apoptosis were analyzed by flow cytometry. The expression of p21WAF1 mRNA was detected by RT-PCR. RESULTS:After treatment with VPA, cell cycle was arrested obviously at G0/G1 phase ［(82.30±9.41)% vs (40.13±2.12)%, P<0.05］. The apoptotic rate was significantly higher in the cells treated with VPA than that in untreated cells ［(11.47%±0.25%) vs (4.77%±0.40%), P<0.05］. The level of p21WAF1 mRNA was increased ［(1.65±0.91) vs (0.25±0.04), P<0.05］. CONCLUSION:VPA induces elevated expression of p21WAF1 mRNA in K562 cells, resulting in G0/G1 phase arrest and apoptosis in vitro.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Effect of atorvastatin on blood pressure in spontaneously hypertensive rats

AIM: To explore the mechanisms underlying the effect of atorvastatin on blood pressure in spontaneously hypertensive rats (SHR). METHODS: The effects of atorvastatin on plasma endothelin-1, aortic nitric oxide synthase, aortic smooth muscle cell (ASMC) apoptosis and p27 expression in SHR were evaluated. 12 eight-week-old SHR were randomized into atorvastatin group (ATV, n=6) and SHR group (n=6). 6 age-matched normotensive Wistar-Kyoto rats (WKY) were served as controls. 50 mg·kg-1·d-1 of atorvastatin was administered to ATV by gavage for 10 weeks. Serum cholesterol and triglycerides were measured, and systolic blood pressure of caudal artery was examined. Plasma endothelin-1 and nitric oxide synthase activity of aortic tissue were measured. ASMC apoptosis rate was detected by TUNEL technique, and positive expression rate of P27 in ASMC was analyzed. RESULTS: After 10 weeks, systolic blood pressure in ATV was significantly lower than that in SHR ［(134.17±3.60）mmHg vs （173.33±3.78）mmHg, P<0.01］. Compared with SHR, serum cholesterol and triglycerides were significantly lower (P<0.01, P<0.01) in ATV. Additionally，atorvastatin significantly decreased plasma endothelin-1 ［(130.04±40.07）ng/L vs （196.74±59.69）ng/L, P<0.05］ and increased nitric oxide synthase activity in aortic tissue ［(0.189±0.040）kU/g protein vs (0.124±0.057)kU/g protein, P<0.01］, compared with SHR. ASMC apoptosis rate was higher in ATV than that in SHR (16.94%±3.08% vs 9.01%±2.36%, P<0.01). Compared with WKY, positive expression rate of p27 in ASMC from ATV was higher (33.02%±5.01% vs 24.25%±4.41%, P<0.05), whereas that was lower in SHR (16.08%±7.09% vs 24.25%±4.41%, P<0.01). CONCLUSION: Atorvastatin may reduce the plasma endothelin-1， up-regulate nitric oxide synthase activity and ASMC P27 expression and facilitate ASMC apoptosis，which may effectively reduce blood pressure in SHR.     

Inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in two human ovarian carcinoma cell lines

AIM: To investigate the inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in human ovarian carcinoma cells (OVCAR8 and SKOV3 cell lines). METHODS: The expression of MISIIR protein and the localization of MISIIR protein were analyzed by Western blotting and confocal spectral microscopy, respectively. Cell apoptosis and cell cycle were detected by flow cytometry (FCM). Cell viability was determined via MTT method. Clone formation test was used to detect oncogenicity in vitro.RESULTS: The MISIIR protein expression in OVCAR8 cells but not in SKOV3 cells was observed. MISIIR expression was seen on the OVCAR8 cell surface and in the cytoplasm with both antibodies. After treated with rhMIS for 48 h, the cell viability was significantly decreased in OVCAR8 cells. rhMIS inhibited the oncogenicity of OVCAR8 cells greatly. The cell apoptosis of OVCAR8 cell exposed to 10 mg/L rhMIS was (31.3±2.1)%, and OVCAR8 cells in the G1 phase were increased by (70.4±3.0)%. Compared to SKOV3 cells the differences were significant (P<0.01). CONCLUSION: Recombinant human Mullerian inhibiting substance suppresses the growth of MISIIR-positive ovarian cancer cells by inducing apoptosis and cell cycle arrest. We predict that rhMIS might be a new target to treat human ovarian malignancies.     

Knockdown of survivin expression using siRNA inhibits the growth of PC-3M cells

AIM: To study the inhibitory effects of survivin siRNAs on the growth of PC-3M cells. METHODS: Two pairs of DNA template coding siRNA against survivin were synthesized to construct two recombinant plasmids, pSi-sur1 and pSi-sur2. The two recombinants and the two controls, lipofectin and vacant plasmid were transfected into PC-3M cells. The expressions of survivin mRNA and protein were detected respectively by RT-PCR and Western blotting. Proliferation abilities were measured by MTT, and the cell cycle and apoptosis were assayed by FCM. RESULTS: After 72 h of transfection, the level of cell survivin mRNA in the two siRNA groups was 48%±6% (n=3) and 30%±5% (n=3) of that in lipofectin group, and expression of survivin protein were 38%±4% (n=3) and 36%±4% (n=3) respectively of that in lipofectin control. The proliferation rate of cells in pSi-sur1 and pSi-sur2 groups was also inhibited according to MTT, about 44.20%±2.08% (n=3) and 39.20%±1.93% (n=3) of that in lipofectin group. Cell numbers of G1 phase in two siRNA groups were significantly higher than that in two controls, while cells of G2 phase and S phase were much lower. Cell apoptosis was found in both siRNA groups. CONCLUSION: The two survivin siRNA significantly inhibit the expression of survivin in mRNA and protein levels, arrest the cell cycle in G1 phase, and suppress the growth of PC-3M cells and induce apoptosis in vitro.     

iASPP on apoptosis in breast cancer cells which expressed wild type p53

AIM: To investigate the RNAi effect of the inhibitory member of the ASPP family (iASPP) on the apoptosis of human breast cancer cell MCF-7 which expressed the wild type p53 gene. METHODS: The recombinant plasmid pAd-iASPP-RNAi was transfected into MCF-7 cells. The expression of iASPP mRNA and protein was analyzed by RT-PCR and Western blotting, respectively. The cell apoptosis was detected by FCM, and then the MCF-7 cells were transplanted into nude mice to set up transplantation model. The expression of iASPP RNA and protein in transplanted neoplasm were determined by RT-PCR and Western blotting, the apoptosis index was detected by FCM at the same time. RESULTS: The results showed that the expression of iASPP descended in MCF-7 cells (mRNA 95.4% and protein 96.8%, respectively, P<0.01) and the apoptosis rate and necrosis rate of MCF-7 cells increased (P<0.01) after transfection. As treated with pAd-iASPP-RNAi, the expression of iASPP in transplantation tumor cells descended 87.4% (mRNA) and 89.2% (protein), respectively (P<0.01), and the apoptosis rate and necrosis rate increased accordingly (P<0.01, P<0.05). CONCLUSION: The inhibition of iASPP may resume the ability of p53 to induce apoptosis in breast cancer cells which is able to express wild type p53.     

A rat model of repeated seizures induced by pentylenetetrazol at different maturational stages and the role of NF-κB in the pathogenesis of epilepsy

AIM: To observe histopathologic changes and NF-κB expression in hippocampus in neonatal and matural rats after repeated seizures, and to explore the role of NF-κB in the pathogenesis of epilepsy in premature brain of rats. METHODS: Neonatal rats and mature rats were divided into 2 experimental groups at 10 days and 60 days after birth (P10 and P60). Convulsions were induced by repeated injection of pentylenetetrazol (PTZ) intraperitoneally for first 5 days. The animals in control group were injected with NS at the same volume in the same conditions. The neurons in CA1, CA3, dentate granule (DG), as well as in hilar were counted by thionin staining, in order to observe the profile of the necrosis and apoptosis. NF-κB expression was examined by immunohistochemistry assay. Timm's method of silver sulfide staining was adopted to observe the mossy fiber sprouting. RESULTS: (1) In immature rats (10 days old), neurons in CA1, CA3 and hilar demonstrated no differences from controls, whereas adult rats (P60) had a significant decrease in number of neurons in CA1 and CA3 (8.22±1.88, 5.62±1.68 vs 6.31±1.50, 3.62±1.40). In adult rats, neurons in dentate granule showed no differences with controls, whereas immature rats with daily seizures had a significant increase (23.25±3.06 vs 16.25±1.58). (2) There was prominent sprouting in the CA3 stratum pyramidal layer in all experimental rats after 5 daily seizures, regardless of the age. However, the degree of sprouting was significantly different between the two experimental groups (3.25±1.03 vs 1.50±0.92, P<0.05). (3) NF-κB was highly expressed in CA3, CA1 and DG after 24 hours by PTZ-kindling, whereas it was little expressed in control group. NF-κB expression was higher in P10 experimental rats than that in P60 rats. CONCLUSION: No cell loss was observed in hippocampus in neonatal rats after recurrent kindling. The high expression of NF-κB may be one of the important molecular mechanisms underlying the special resistance of the neurons in premature brain to the epileptic cerebral lesions.     

Effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro

AIM: To observe the effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro. METHODS: The lymphocytes from the lymphoid nodes of BALB/c mice were isolated and primarily cultured. The viability of T cells was assessed by MTT assay. Fluorescence-conjugated monoclonal antibody and flow cytometry (FCM) were used to analyze the expression of T-cell activation marker CD69 in response to concanavalin A (Con A) in vitro. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to detect the proliferation of T cells in vitro. FCM analysis was used to determine the production of reactive oxygen species (ROS) in the T cells by staining with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). The mean fluorescence intensity of DiOC₆(3) staining in the T cells was detected by FCM in order to analyze the effects of salidroside on the activity of the mitochondrial and the mitochondrial membrane potential in the T cells induced by dexamethasone (DEX). The thymus T cells from BALB/c mice were isolated and primarily cultured, and then FCM was also used to analyze the apoptosis of the thymus T cells treated with DEX. RESULTS: Salidroside increased the expression of T-cell activation marker CD69 at the final concentration of 80, 160 and 320 μmol/L (P<0.05). Salidroside promoted the proliferation of T cells induced by Con A for 72 h in vitro (P<0.01). Salidroside reduced the production of ROS (P<0.05) and protected the mitochondrial membrane potential of T cells from the injury of DEX (P<0.01). Salidroside also decreased the apoptosis rate of the thymus T cells induced by DEX in vitro (P<0.01). CONCLUSION: Salidroside promotes the activation and proliferation of T cells induced by Con A, reduces the production of ROS, maintains the mitochondrial membrane potential and protects thymus T cells against apoptosis induced by DEX in vitro.     

Role of Kv1.3 channel of CD4+T lymphocytes in the formation of atherosclerosis in rat spleen

AIM: To investigate the function of voltage-gated potassium channel Kv1.3 and its possible role in CD4⁺ T lymphocytes in the formation of atherosclerosis (AS) in rat spleen. METHODS: The rat atherosclerosis model was established by feeding high-fat diet. The proportion of lymphocytes was determined by flow cytometry. The CD4⁺ T lymphocytes were separated using immunomagnetic bead. The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was detected. The concentrations of intracellular calcium and cytokines were also measured. RESULTS: (1) The proportion of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (74.93%±2.15% vs 67.80%±2.54%, P<0.05). (2) After stimulated with concanavalin A (ConA), the proliferation of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (1.1321±0.1750 vs 0.7971±0.0955, P<0.05). (3) After stimulated with ConA, the concentration of intracellular calcium in AS group was higher than that in control group. (4) In AS group, the releases of cytokines of IL-2 and TNF-α in AS group were significantly higher when stimulated with ConA for 48 h than that for 24 h. (5) The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was greatly higher in AS group than that in control group (3.670±1.579 vs 1). CONCLUSION: In AS rats, the increase in CD4⁺ T lymphocytes as well as the augmentation of Kv1.3 mRNA expression in the cells suggest that up-regulation of Kv1.3 mRNA expression in CD4⁺ T lymphocytes may be involved in the mechanism of atherosclerotic formation in rat spleen.