Background Posterior capsular opacification (PCO) is a primary cause of blurred vision after extra-capsular cataract extraction (ECCE),and there is a higher incidence of PCO in the patients with diabetes mellitus.Echistatin (Ecs) can suppress the proliferation of lens epithelial cells (LECs) and thereby inhibit the formation of PCO.However,its mechanism and safe dose deserve to study.Objective The aim of this study was to observe the inhibitory effect of different concentrations of Ecs on LECs proliferation in the early stage of PCO in diabetic rabbits and explore the safe dose of Ecs.Methods Diabetic mellitus was induced by injection of 90 mg/kg alloxan via ear vein in 15 New Zealand white rabbis.ECCE was performed in the right eyes of the rabbits.The rabbits were randomized to the control group and 5.0,7.5,10.0 and 15.0 μg/ml Ecs group according to randomized number table method.Ecs of 0.2 ml in above doses was injected into the anterior chamber after ECCE in different concentrations of Ecs groups,and 0.2 ml distilled water was used in the same way in the only diabetic rabbits as the control group.Postopeartive response of ocular anterior segment was observed and PCO was graded under the slit lamp microscope.The corneal and retinal specimens were prepared 10 days after operation for the assay of preliferative cell nuclear antigen (PCNA) expression in LECs by immunochemistry to evaluate the effective dose of Ecs.Regular histopathological examination was performed,and apoptosis of corneal endothelial cells and retinal cells was detected by TUNEL method to assess the safe concentration of Ecs.Results Different degrees of corneal edema and exudation in anterior chamber were seen in the eyes of different groups.The inflammatory response disappeared 3-5 days in the control group and 5.0 μg/ml Ecs group and 7 days in the ≥7.5 μg/ml Ecs groups.PCO was 1-2 grade in the control group and 5.0 μg/ml Ecs group and 0 grade in the ≥ 7.5 μg/ml Ecs groups.The difference in the positive expression level (absorbance,A) for PCNA in LECs was significantly different among the control group and various Ecs groups (F=18.006,P=0.001),and the positive expression level of PCNA in the ≥ 7.5 μg/ml Ecs groups was markedly reduced in comparison with that in the control group (P =0.010,0.001).Hematoxylin and eosin staining showed an normal morphology and order arrangement in corneal endothelial cells and intact structure in retinal internal limiting membrane in the groups.TUNEL assay revealed that the apoptosis values (mean A value) of corneal endothelial ceils and retinal cells in the ≤ 10.0 μg/ml Ecs groups were not significantly changed in comparison with the control group (all at P＞0.05),but the apoptosis values in the 15.0 μg/ml Ecs group were markedly higher than those in the control group (P=0.004,0.018).Conclusions Ecs can inhibit the early PCO in diabetic rabbits and show the optimal effect at the concentrations of 7.5 and 10.0 μg/ml without visible eytotoxicity to eye other tissues.Therefore,these two doses of Ees might be used for the study of long-term therapeutic effectiveness.

Objective: To investigate the immunotherapeutic effects of elemene combo tumor cell vaccine(EC TCV) on Hca F murine hepatic carcinoma and changes of IL 10 and IL 12 secretion by splenocytes of treated mice. Methods: BALB/c mice inoculated with Hca F were treated with EC TCV, CY, EC TCV+CY or PBS control, tumor growth was measured, concentration of IL 10 and IL 12 in supernatants of mixed splenocytes and Hca F culture(MSTC) was assayed with ELISA. Results: After EC TCV immunotherapy and CY chemotherapy, the latent period of tumor growth prolonged, mean tumor weight (MTW) decreased, even no nodule was observed in EC TCV+CY chemoimmunotherapy group, the concentration of IL 10 in supernatants of MSTC of EC TCV immunotherapy group decreased significantly(1.82?0.29 pg/ml, P

Objective:To find out the role of JNK/SAPK signaling-transduction pathway in the effect of elemene against hepatocarcinoma, offering the clue to ilustrate the molecular mechanisms of antitumor effects of elemene. Methods: The detection of the distribution of elemene in Hca-F cells was detected by gas chomatography and apoptotic changes in elemene treated. SMMC7721 cells were examined by TEM. After elemene treatment, the activation of JNK/SAPK in HepG2 cellls and the DAXX gene expression in SMMC7721 cells were detected by Western blotting and RT-PCR respectively. Results: Gas chomatography showed that elemene was detected at 8. 42 minute. The SMMMC7721 cells treated by elemene for 3 hours began to show typical apoptotic changes . The JNK/SAPK activity in HepG2 cell treated with heat shock was the highest of all groups and the group treated with elemene was the next and the control group is the lowest one. There was no DAXX gene expression in SMMC7721 cells treated with elemene. Conclusion: Elemene can diffuse into cells. Tumor cell apoptosis treated with elemene may be induced by JNK/SAPK activating and DAXX signal pathway may not play key role in JNK/SAPK activation induced by elemene.

Objective:Normal murine DCs were pulsed with complex of tumor antigen from elemene-combo tumor cell vaccine-heat shock protein 70 of BCG (H TA-HSP70 BCG).Their proliferation and antigen presenting function were evaluated.Methods:The dendritic cells(DCs)were cultured in complete media containing GM-CSF and IL-4 and pulsed with H TA-HSP70 BCG,H TA or HSP70 BCG.Their proliferation and stimulating effects on spleen nonadherent cells were evaluated with MTT assay.Their capability of endocytosing FITC labeled dextran was assayed with FACscan,morphological changes of DC were observed in electron microscope.Results:Proliferation index of DCs pulsed with H TA-HSP70 BCG was 2.107?0.013,proliferation index of DCs pulsed with H TA-HSP70 BCG mixed with normal nonadherent spleen cell was 1.927?0.073.The percent of DCs endocytosed FITC labeled dextran was 58.61%.Above changes were more significant than those of DCs pulsed with H TA or HPS79 BCG.Conclusion:H TA-HPS70 BCG had more potent activity to pulse DC and strengthen antigen presenting of DC.

In this experiment, Hca-F or L615 elemene combo-TCV (H-TCV and L-TCV), H-TCV lysates, corynebacterium parvum (CP) were used to immunize 615 and Balb/c inbred mice, and their splenic DCs were prepared and pulsed in vitro with tumor cell lysates (H or L) and TCV lysates (TH or TL). The capacities of DCs to stimulate the proliferation of syngeneic nonadherent spleenic cells were tested with MTT assay. The results showed that the splenic DCs from normal mice in vitro pulsed with TH or TL could induced syngeneic noradherent splenic cells to proliferate (P＜0.01), while the H or L pulsed DCs could not. The splenic DCs from H-TCV or L-TCV or TH immunized mice re-pulsed in vitro with TH or TL exhibited stronger stimulating effects than the DCs from normal mice pulsed in vitro for the firth time pulsed with TH or TL (P＜0.01 or P＜0.05); The capacities of DCs to induce proliferation of syngeneic nonadherent splenic cells could be further enhanced by CP immunization, especially when were pulsed with TH (P＜0.01). Normal inbred 615 mice were transferred with DCs pulsed with lysates of elemene TCV (TDCs) or pulsed with lysates of Hca-F tumor cells (HDCs) on day 7 before challenged with lethal dose of live Hca-F cells, significant adoptive immunoprotective effects were seen, with 61.6% tumor inhibition rate and 25% survival in TDC adoptive transfer group. This study indicated that DCs might play a role in the mechanisms of active immunization and pulsing DCs with lysate of elemene combo TCV and isolating DCs from elemene combo TCV immunized mice were useful methods for DCs vaccine preparation.

Elemene is a new anticancer drug isolated from a Chinese traditional medicine Curcuma aromatica. In previous work, we discovered that tumor cell vaccine (TCV) treated with oleum Curcuma aromatica or elemene could induce significant immunoprophylactic effect against a variety of aminal tumor strains and the method of preparation of elemene combo-TCV(EC-TCV) already got China's inventive patent. In this paper we further studied the active immunotherapeutic effect and the possible cellular/molecular mechanisms of EC-TCV immunization. The results were as follows:(1) EC-TCV immunization showed significant therapeutic effects (P＜0.05) against murine Ca761 syngeneic mammary carcinoma (H-2k) and HCa-F allogeneic hepatic carcinoma (H-2-) models; (2) The spleen cells of Hca-F EC-TCV immunized mice displayed higher cytotoxicity and IL-12 level while the secretion of IL-10 was decreased (P＜0.05); (3) Similar to heat shock, elemene(E), mitomycin C(MMC) and glutaraldehyde (G) could act alone as stressor, and induce significant changes of the expression of membrane heat shock proteins(HSP70 or/and HSP90) on L615 leukemia and HCa-F hepatoma cells and the EC-TCVs (E+MMC+G treated in combination) showed the highest level of membrane HSPs expression (P＜0.05 or P＜0.01 );(4) The HSP70-peptide complex isolated from HCa-F EC-TCV through ADP-agarose affinity chromatographic system could induce active immunoprotection against lethal dose challenge of HCa-F hepatic cancer cell but could not protect against the cross challenge of lethal dose of L615 leukemia. The results indicated that the immunoprotective effect of EC-TCV was in some extent tumor-specific, MHC-nonrestricted, and HSPs might play an important role in its molecular mechanisms.

OBJECTIVEIn vitro model of hydrogen peroxide induced apoptosis of SW-480 cells was used to investigate the role of NF-kappaB in the pathogenesis of reactive oxygen species induced apoptosis of intestinal epithelial cells.

METHODSUltra-structural changes were observed. Apoptosis of SW-480 cell line was determined by Annexin-V and PI double-stained flow cytometry. Nuclear translocation of NF-kappaB was determined by anti-NF-kappaB polyclonal antibody and EB double-staining. NF-kappaB activity was studied by electrophoretic mobility shift assays. RT-PCR was performed to study expression of NF-kappaB mRNA.

RESULTSHydrogen peroxide led to apoptosis of SW-480 cells, condensed or semilunar chromatin even apoptotic bodies could be observed. Nuclear translocation of NF-kappaB, increase of NF-kappaB activity and expression of NF-kappaB mRNA were found simultaneously.

CONCLUSIONSEarly activation of NF-kappaB may be one of the mechanisms of apoptosis in intestinal epithelial cells by reactive oxygen species.

Apoptosis ; Humans ; Hydrogen Peroxide ; toxicity ; Intestinal Mucosa ; metabolism ; Microscopy, Confocal ; NF-kappa B ; metabolism ; Reactive Oxygen Species ; toxicity ; Tumor Cells, Cultured

OBJECTIVETo explore the activation of polymorphonuclear cells (PMNs) and vascular endothelial cells co-cultured with and stimulated by lipopolysaccharide.

METHODSPMNs in concentration of 2 x 10(6)/ml isolated from healthy volunteers by Percoll gradient were added to monolayer of ECV-304 cells grown to confluency, then different groups were prepared according to final concentration of lipopolysaccharide. The morphological change was observed under invert microscope. The changes in TNFalpha and IL-6 levels of the supernatant of the cultured cells were determined at 4, 8, 12 and 24 hours after culturation.

RESULTSThe TNFalpha production of cultured pure ECV-304 exhibited no remarkable change when stimulated by different concentrations of LPS. But the TNFalpha production of the ECV-304 increased significantly when co-cultured with PMNs at 4 hr and stimulated by LPS in concentration of 10 micro g/ml, and increased at 8 hours and lasted up to 24 hours of culturation in higher levels (P < 0.05). The IL-6 production of cultured pure ECV-304 increased obviously along with the increase of LPS concentration, and it showed no change when PMNs co-cultured with ECV-304. While the IL-6 level in the supernatant of co-cultured ECV-304 with PMNs increased sharply when stimulated by both low (100 ng/ml) and high (1 micro g/ml) concentrations of LPS and maintained at high levels up to 24 hours of culturation. The higher the concentration of LPS was, the quicker the IL-6 level increased.

CONCLUSIONCo-cultured PMNs and endothelial cells could be activated and activation state could be maitained by low concentrations of LPS.

Adult ; Cell Line ; Cells, Cultured ; Coculture Techniques ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Neutrophils ; cytology ; drug effects ; metabolism ; Time Factors ; Tumor Necrosis Factor-alpha ; metabolism