Effects Of Brucine On Secretion Of T Lymphocytes And Expression Of T-bet In Patients With Aplastic Anemia

Effects of brucine on secretion of T lymphocytes and expression of T-bet in patients with aplastic anemiaObjectiveBy observing the effects of brucine on levels of TNF-α,IFN-γ,IL-4,were secreted by peripheral blood T lymphocytes and expression of T-bet in patients with aplastic anemia, to investigate the mechanism of nux vomica in the treatment for aplastic anemia.MethodPeripheral blood T lymphocytes were isolated, purified from 10 patients with aplastic anemia and 10 healthy volunteers. Collected 10 ml Peripheral blood and separated T lymphocytes by density gradient centrifugation method and immunomagnetic beads. Variable count and purity measurement by flow cytometry were necessary to further research (>95%). T lymphocytes from healthy volunteers were normal control groups and no interference. T lymphocytes from patients were experimental groups, were divided into 4 groups randomly, black group, 100ug/ml,200ug/ml and 400ug/ml brucine treated group (100ug/ml PHA was added to all groups) respectively. After cultured for 72 hours, the levels of TNF-a, IFN-y, IL-4 in the culture supernatant were detected by mean of Enzyme-linked immunosorbant assay (ELISA); and collected T lymphocytes and abstracted RNA in all groups. The expression levels of T-bet were detected by mean of Reverse transcription-polymerase chain reaction (RT-PCR).Results1. Compared with normal control, the levels of TNF-α(p<0.01) and IFN-γ(p<0.01) in the culture supernatant of blank group were significantly increased, and IL-4 were significantly decreased (p<0.01). The expression level of T-bet in blank group was higher than that of normal control (p<0.01).2. The levels of TNF-αand IFN-γin brucine treated groups were lower than blank group, and depended on concentration of brucine; The difference among brucine treated groups were significant. Both of them in 400ug/ml group were same as that of normal control.3. The levels of IL-4 were invariable in brucine treated groups (p>0.05).4. Compared with normal control, the expression levels of T-bet in 100ug/ml (p<0.01), 200ug/ml (p<0.01) and 400ug/ml group (p<0.01) were significantly decreased; 200ug/ml (p<0.01) and 400ug/ml group (p<0.01) were lower than 100ug/ml group; The difference among normal control,200ug/ml and 400ug/ml group was no significant.5. In experimental groups, the expression levels of T-bet were positive correlation with the levels of IFN-y(r=0.795, p<0.01).Conclusion1. The levels of TNF-a, IFN-y in patients with aplastic anemia were higher than healthy person, but the levels of IL-4 were lower.2. The level of T-bet mRNA in patients with aplastic anemia was higher than healthy person.2. Brucine could reduce the levels of negative regulation factors TNF-a and IFN-y and the expression of T-bet in patients with aplastic anemia, and the effects depended on concentration of brucine, and the level of IFN-y maybe mainly regulated by T-bet.3. Above that was not only the evidence for brucine in the treatment for aplastic anemia, but also the scientific evidence for use of nux vomica in clinic.