| BACKGROUND:Gelsemium elegans Benth(GEB) is the whole grass of the Loganiacae plant Graceful Jesamine. The drug is warm in nature and pungent in flavour. It is a traditional medicine in our country and is also famous of its poisonous all over the world. "Compendium of Materia Medica" recorded that GEB has the effect of anti-spasm, analgesia and diuresis. In our country, folks of Fukien, Guangdong, Guangxi had used GEB to cure the rheumatism, the sore and carbuncle, injury from fall and other skin diseases. In recent years, studies had discovered that the GEB has various pharmacology functions, such as anti-respiratory center and anti-heart contraction, relaxing smooth muscles, analgesia, mydriasis, inducing the apoptosis of tumor cells, promoting phago-functions of macrophages, anti-platelet aggregation, etc. H Gerrad had made it into green perparation in 1887. As a legal product, it was accepted by the vice- pharmacopeia of many nations; at the same time, it was recorded by American medicine index and "The Civil Medicine of The World" in Japan.Since the 70's in 19 centuries, American researcher T. G Wormley had started the investigation about the components of GEB, and then he obtained a monomer. The local scholars began to learn the pharmacognosy, chemistry, toxicology arid pharmacology, etc. of GEB since the 30's in last century. Because of its toxicity, and its approach between therapy dosage and intoxication dosage, GEB is limited to be extensively used in clinic. GEB mainly contains indole alkaloid, the various classes and contents of alkaloid in the plant body depend on the different places of production. Koumine (Kou) is the highest content component of GEB from China. The toxicity of Koumine could not represent the toxicity of original plant. Studies showed that the toxicity of Koumine is lower than Gelseminum total alkaloids, and in mouse the ip LD50 is about to 1/50 of Gelseminum total alkaloids. It suggests that Koumine may be the active component of GEB. Study on the mechanism of Koumine on analgesia, anti-tumor, immunoloregulation and anti-psoriasis, etc. will provide us the experiment basis for further developing Koumine as the safe clinical product.The psoriasis is a common, chronic, recurrent, inflammatory proliferative epidermal skin disease. Its incidence rate in population is 0.1%~3%. There are various cells, such as epithelial cells, T cells, monocyte, histocyte, endothelial cells and nerve cells, etc. which participate the process of lesion in complicated pathogenesis of the psoriasis. Keratinocytes was thought as the key cells of psoriasis until 1986, when Valdimarsson and his research group suggested that local infiltrated activated T cells lead to anomaly proliferation of keratinocytes. T cell was gradually known as an important role in episode of psoriasis. Studies have suggested that T cells are effector cells within the psoriasis episode process. Interaction of the T cells and the epithelial cells was known as the most important procedure. The immunology mechanism of psoriasis is that MHC- II DR+ antigen-presenting cellspresent the ectogenesis antigen to the epidermis CD4*T lymphocytes, which cause the disorder of cell regulation and lead to the transformation of phenotype and function of CD4+ T cells. The activated CD4+ T cells can release the cytokines such as IL-2, EL-6, IL-8, IFN- y etc. These cytokines can stimulate the proliferation and abnormal differentiation of epithelial stem cells. Along with further studies of episode process, the targeting drug systems for psoriasis have become a focal point.In recent years, studies indicate that some Chinese medical herbs or their extracts, such as common threewingnut root, could be used in treatment of psoriasis. These suggested that the traditional Chinese medicine may have potential purpose of psoriasis. Our lab extracted Koumine crystal from Gelsemium elegans Benth and observed the effect of Koumine on general behavior in experimental animal psoriasis models. Higher and medium dosage groups of Kou showed a remarkable inhibiting effect on mouse vaginal epithelial mitosis and promoting effect on the formation of epidermal granular layer in the scales of mouse-tail. Kou has certain anti- psoriasis activity. Kou can also decrease the level of IL-2 in serum, repress the high cell immunity and improving the immunity state. But the anti-psoriasis mechanism of Kou is not yet clear.PURPOSE:From the point of theimmunopathogensis and immunities therapeutics of psoriasis, we observe the effect of Koumine on CD4*T lymphocytes separated from murine spleen cells. We study and analyse the effect of Koumine on cell function, cytokines levels and calcium signaling during T cell activation, in order to elucidate the anti-psoriasis and immunoloregulation certain mechanism of Kou and to provide evidences for Kou in development of clinical preparation.METHODS:The CIX'T lymphocytes were isolated from murine spleen cells by Magnetic activated cell sorter (MACS) with biotin-antibody cocktail and anti-biotin microbeads. Fluorescent activated cell sortor (FACS) was used to analyze the purity of CD4+ T cells. Cell vitality was detected by trypan blue.The CD4+T lymphocytes were isolated by MACS and stimulated by mitogen(ConA or PHA) for 72h in the presence or absence of Kou(10~320ug/ml). Cell proliferation was assessed by MTT. CD4+T lymphocytes were stained by propidine iodide (PI).Cell cycle were detected using FACS.Cells were stimulated by mitogen, different concentration Kou test group supernatant of culture cells was collected at different time. The expression of Thl type cytokine IL-2> IFN-y and Th2 type cytokine BL-4> IL-10 in supernatant of culture cells were assayed by enzyme-linked immunoabsorption assay (ELISA).Laser scanning confocal microscope (LSCM) of the calcium fluorescent indicator dye Fluo-3/AM were used to determine the [Ca2+]i in murine CD4+T cells induced by Kou.RESULT AND CONCLUSION:1. The purity of CD4+T cells analyzed by FACS attained to (90.43±l .93)% after magnetic separation. Cell vitality detected by trypan blue was (94.9±3.6)%. MACS is an simple and high efficiency method for high purity and vitality cells. CD4*T cells derived from murine spleen can be purified by MACS, and they can survive and differentiate after time.2. After magnetic separation, CD4+T cells showed normal proliferation induced by ConA and PHA. Kou (40~320ng/ml) show significant anti-proliferation effecton CD4+T cells. Stimulation index (SI) of ConA and PHA on CD4+T cells showed a dose-dependent inhibition by Kou.3. Compared with ConA control group, lOOug/ml Kou exerted significantly effect on cell cycle after 24 hours. The result showed that G0/G1 phase cells rise from 52.2% to 74.0%, the S phase cells decreased from 43.3% to 25.4%. The cell cycle of CD4+T cells was blocked in G0/G1 phase after treatment with Kou. This supposed that Kou could regulate the cell cycle and interference DNA synthesis of CD4+T cells.4. Excertion level of Thl type cytokine IL-2 and EFN-y in CD4+T cells induced by 5ug/ml ConA could be significantly decreased by Kou with time and dose-dependent. On the other hand, Kou slightly increased IL-4 level in supernatant of culture cells, but this effect has no statistics difference. Compared with control group, Kou increased the low level of IL-10 in supernatant of culture cells in first 24h. After that, 20 ^ 200ug/ml Kou decrease the high level of IL-10. But, Kou (lOOug/ml) increase the EL-10 level which indicated that Kou (lOOug/ml) may promote the recovery of T cell immunological function. These results suppose Kou may accelerate the differentiation of CD4+T cells stimulated by mitogen to Th2 cells. Kou may regulate the cell immunological function by decreasing Thl cytokin level and increasing Th2 cytokin level.5. At the resting state in HBSS, Kou(100ug/ml) has no effect on [Ca2+]i in murine CD4+T cells, but significantly decreased [Ca2+]; in murine CD4*T cells activiated by mitogen. This indicated Kou may refrain extracellular Ca2+ influx in activated CD4+T cells. Kou(100ug/ml) decreased significantly [Ca2+]i in cellsin the absence of Ca2+ in the media after treatment with heparin and caffeine. This indicated Kou may block store-operated Ca2+ channel by refrain Ryanodine receptors (RyR).In short, CD4+T lymphocytes isolated by MACS was used as experiment model to investigate pharmacology mechanism of Kou on immunity disease. Kou inhibit proliferation of CD4+T cells in vitro. Its anti-psoriasis function may be due to that Kou blocked CRAC on active cell membrane and blocked store-operated Ca2+ channel by refrain RyR. Further more, Kou interfered the Th cell cytokine production through decreasing IL-2> IFN- Y level and increasing IL-10 level in supernatant of culture cells. |
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