Antitumor activity of extracts from rabbit skins inflamed by Viccinia virus vaccine in vitro / 国际药学研究杂志

Objective To investigate the effect of extracts from rabbit skins inflamed by Viccinia virus vaccine (analgecine) on the proliferation of human cancer cells and on the cytokine secretion in mouse spleen lymphocytes in vitro. Methods Five human tumor cell lines, HepG2, LM3, H460, A549 and HeLa were used and the effect of analgecine (1.63, 0.815, 0.326, 0.163 and0.0815 U/ml) on the cell proliferation was evaluated by the CCK-8 assay. The mouse spleen was isolated aseptically, and the spleen lymphocyte suspension was prepared and cultured with PRMI-1640 medium containing 10％ fetal bovine serum (FBS). For detection of the cytokine IL-2, IFN-γ, IL-4 and IL-12 level, the stimulant concanavalin A (ConA) or lipoplysaccharide (LPS) was added into the lymphocyte suspension, and the lymphocytes were cultured under the presence of analgecine at the final concentration of 0.815, 0.163 and 0.0815 U/ml for 24 hours. Then, the level of the cytokines in the supernatant was detected by the ELISA kit. On the other hand, the effect of supernatant of the spleen lymphocyte cultures under the presence of analgecine at 0.815 U/ml on the proliferation HepG2 cells was also evaluated by the CCK-8 assay. The CCK-8 assay was performed after cultivation of the HepG2 cells in the whole supernatant or in its dilution with fresh medium for 24 hours. Results Analgecine showed a dose-dependent inhibitory effect on the five tested cancer cell lines, with the inhibition rate of 58.95％, 55.08％, 57.28％, 45.80％ and 48.18％ at the 1.63 U/ml on the HepG2, LM3, H460, A549 and HeLa cells, respectively. Compared with the control group, the secretion of IL-2, IFN-γ and IL-4 was significantly increased in the 0.163 and 0.815 U/ml analgecine groups (P＜0.01), while the secretion level of IL-12 was increased in the 0.0815, 0.163 and 0.815 U/ml analgecine groups (P＜0.01). The supernatant of the mouse spleen lymphocyte cultures under the presence of0.163 U/ml analgecine could inhibit the HepG2 cell proliferation in a dose-dependent manner, and the inhibitory effect of the whole supernatant was significantly stronger than the effect of the same concentration analgecine 0.163 U/ml (P＜0.01). Conclusion Analgecine could inhibit the cell proliferation of the tested five human cancer cell lines, increased the secretion of IL-2, IFN-γ, IL-4 and IL-12 cytokines in mouse spleen lymphocytes, all in vitro, and its effect on the cytokine secretion may be related to the inhibitory effect on the human cancer cell proliferation.

Effects of Polysaccharides from Millettia speciosa Champ on Proliferation of Spleen Lymphocyte and Secretion of Cytokine in Mice / 医药导报

Objective To explore the mechanism of immunoregulation by investigating the effects of polysaccharides from Millettia speciosa Champ (MSC) on proliferation of spleen lymphocyte and secretion of cytokine in mice.Methods The effects of MSC polysaccharides on Con A-induced spleen T lymphocyte proliferation were determined by MTT.The contents of TNF-α, IL-6 and PGE2 were determined by ELISA.Results The Con A-induced T lymphocyte proliferation was significantly increased by MSC polysaccharides at the concentrations from 50 to 200 μg·mL-1.Coupled with TNF-α and IL-6 were significantly increased while PGE2 was significantly decreased.Conclusion MSC polysaccharides could increase proliferation of spleen lymphocyte and enhance the immune responses by increasing the secretion of TNF-α and IL-6 in mice.

Prediction and identification of T-cell epitopes in major group 3 allergen de-rived from Dermatophagoides farina / 中国血吸虫病防治杂志

Objective To predict and identify T cell epitopes of major group 3 allergen derived from Dermatophagoides fari-na(Der f 3). Methods The T cell epitopes of Der f 3 were analyzed through the sequence analysis by using the bioinformatics online tools. The five predicted peptides of T-cell epitopes were artificially synthesized. The spleen lymphocytes were co-cultured with the five T cell epitopes by using the modified MTT method and the levels of IL-2,IFN-γ,IL-4 and IL-5 in the supernatant of the cultures were detected by ELISA. Results Five T cell epitopes of Der f 3 were predicted and three of which could pro-mote the proliferation of the mouse spleen lymphocytes. The secretions of IL-2 and IFN-γwere significantly induced and the se-cretions of IL-4 and IL-5 were significantly decreased by three of five prediction epitopes of Der f 3:37GDCPYQISLQSSSHFC-GG54,98IYQHENYDSMTIDNDVALIKLKTPMT123 and 164SELQRVDIDVVSREQCDQLYS184. Conclusion Three T cell epitopes of Der f 3 have been initially identified,which lays the foundation of the diagnosis and treatment of asthma.

Immunomodulatory effect of Prepared Radix Rehmanniae extract in vitro / 中国药学杂志

OBJECTIVE: To investigate the active substances with immunoenhancement effect in Prepared Radix Rehmanniae and its mechanism of action. METHODS: Balb/c mice lymphocytes were isolated from thymus and spleens with conventional methods. MTT assay was carried out for proliferation detection, and then ELISA assay was used to measure the levels of Th1 cytokines IL-2, IFN-γ and Th2 cytokines IL-4, IL-5 of lymphocytes from thymus and spleens of the mice. RESULTS: The water extract (0.049, 0.49 and 4. 9 mg · mL-1) and crude polysaccharide extract (0.032, 0.32 and 3.2 mg · mL-1) of Prepared Radix Rehmanniae resulted in strong promotion of thymus and spleen lymphocytes proliferation and obvious increase of IL-2, IFN-γ, IL-4 and IL-5 levels with or without Con A stimulation. These effects were dose-related. CONCLUSION: Prepared Radix Rehmanniae can significantly enhance immune responses, and the active substance might be crude polysaccharide. The mechanism of their immunoenhancement effects is partly due to enhancement of gene expression of Th1/Th2 cytokines of T cells.

High susceptibility of activated lymphocytes to oxidative stress-induced cell death

The present study provides evidence that activated spleen lymphocytes from Walker 256 tumor bearing rats are more susceptible than controls to tert-butyl hydroperoxide (t-BOOH)-induced necrotic cell death in vitro. The iron chelator and antioxidant deferoxamine, the intracellular Ca2+ chelator BAPTA, the L-type Ca2+ channel antagonist nifedipine or the mitochondrial permeability transition inhibitor cyclosporin A, but not the calcineurin inhibitor FK-506, render control and activated lymphocytes equally resistant to the toxic effects of t-BOOH. Incubation of activated lymphocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. These results indicate that the higher cytosolic Ca2+ level in activated lymphocytes increases their susceptibility to oxidative stress-induced cell death in a mechanism involving the participation of mitochondrial permeability transition.

O presente estudo demonstra que linfócitos ativados de baço de ratos portadores do tumor de Walker 256 são mais susceptíveis à morte celular necrótica induzida por tert-butil hidroperóxido (t-BOOH) in vitro quando comparados aos controles. O quelante de ferro e antioxidante deferoxamina, o quelante intracelular de Ca2+ BAPTA, o antagonista de canal de Ca2+ nifedipina ou o inibidor da transição de permeabilidade mitocondrial ciclosporina-A, mas não o inibidor de calcineurina FK-506, inibiram de maneira similar a morte celular induzida por t-BOOH em linfócitos ativados e controles. Os linfócitos ativados apresentaram redução do potencial de membrana mitocondrial induzida por t-BOOH num mecanismo sensível a ciclosporina-A. Nossos resultados indicam que o aumento da concentração de Ca2+ citosólico em linfócitos ativados aumenta a susceptibilidade dos mesmos à morte celular induzida por estresse oxidativo, num mecanismo envolvendo a participação do poro de transição de permeabilidade mitocondrial.

The Effects of Akaloid Fraction of Korean Ginseng on the Radiation-Induced DNA Strand Breaks / 대한치료방사선과학회지

PURPOSE: To investigate the effect of alkaloid fraction from Korean ginseng on radiation-induced DNA double strand breaks(dsb) formation and repair in murine lymphocytes. MATERIALS AND METHODS: We used the neutral filter elution technique to assay 60 Co gamma ray-induced DNA double strand breaks formation and repair in C57BL/6 mouse spleen lymphocytes for evaluation the dose-response relationship in the presence of alkaloid fraction as a radioprotective agent. The lymphocytes were stimulated with phytohemagglutinin (PHA, 2 mug/ml) to label 3 [H]-thymidine. Isotope-labelled lymphocytes in suspension were exposed to 100 Gy at 0degree C in the alkaloid fraction-treated group and elution procedure was performed at pH 9.6. The extents of formation of radiation-induced DNA double strand breaks and repair were compared respectively via strand scission factor (SSF) and relative strand scission factor (RSSF). RESULTS: Alkaloid fraction reduced the formation of double strand breaks with dose modification factor of 2.15, compared to control group. Rejoining of DNA dsb appeared to take place via two components. The first fast component was completed within 20.4 minutes, but the second slow component was not completed until 220.2 minutes after irradiation. About 30% of dsb formed by irradiation was ultimately unrejoined despite the administration of alkaloid fraction. The administration of alakaloid fraction had a great effect on the second slow component of repair ; the half-time of fast component repair was not changed, but that of slow component was 621.8 minutes. CONCLUSION: Neutral filter elution assay proved to be a very effective method to quantitate the extents of DNA dsb formation and its repair. By using this technique, we were able to evaluate the efficiency of alkaloid fraction from Korean ginseng as a valuable radioprotector. Alkaloid fraction can be used prophylactically to prevent or ameliorate the severe radiation damages in workers and neighbors aound the atomic power plants. For more refined study, however, more advanced purification of alkaloid fraction will be neended in the near future.

Cell Mediated Immunity after Burn—Blast Combined Injuries in Rats and Regulatory Effects of Exogenous Indomethacin and PGE_1 / 中华创伤杂志

This experiment was taken to study the changes of cell mediated immunity (CMT) after burn—blast combined injuries in rats and the regulatory effects of indomethacin and PGE_1 on CMT. Mitogen stimulated blastogenic transformation (MSBT) and interleukin—2 (IL—2) were measured in 54 rats before injury and on 1, 3, 5, 7, 14 days after injury. The results showed that MSBT increased significantly on days 3. 5 and 7 but decreased significantly on day 1. IL—2 increased significantly after injury and did not return to normal until day 14. MSBT and IL—2 production were significantly depressed by PGE_1 but on the other hand much enhanced by indomethacin. PGE_1 and indomethacin had no effect on MSBT and IL—2 production in the control group. These results indicate: 1. moderate burn—blast combined injuries can stimulate CMI in rats: 2. PGE_1 can significantly depress IL—2 production and MSBT in rats after injury: 3. Indomethacin can enhance IL—2 production in rats after injury and 4. PGE_1 and indomethacin have no regulatory effect on IL—2 production and MSBT in normal rats.