| ObjectThe psoriasis-associated antigen Pso p27, which was isolated from patients of psoriasis by Prof. Iverson, is shown to be a major antigen in the immune reactions in psoriasis lesions. It can be produced by mast cells and expressed in the skin lesions, and when the lesions are in remission, the expression of the antigen is reduced or omitted. What is more, Its immune complex can be detected in peripheral blood of psoriatic patients.Traditional Chinese Medicine (TCM) made a great effect in treating psoriasis. And we have proved it can influence the expression of Pso P27. There was a suppression of Pso P27after treatment with TCM. So, we want to know the exactly mechanism of Pso p27participated in the pathogenesis of psoriasis and if the Pso p27play a crucial role in the mechanism of TCM in treating psoriasis in this study.ContentThe content can be divided into2parts.(1)We detected the influence of pso p27antigen on the proliferation and the release of cytokines of psoriasis associated immunological cells (T cell and PBMC cell), and the proliferation of Hacat cells(a cell line of keratinocyte). And we made Qingre Liangxue Jiedu Decoction drug serum to observe the function of TCM on the above content. Here are the details of the experiments:we cultured T cells and PBMC cells which were separated from the psoriatic patients and Hacat cells. And then, we add pso p27in the culture medium of them for24h to detecte the proliferation of T cells and PBMC cells cells by WST-1and WST-8assay, and the release of cytokines by ELISA assy. What is more, we used the supernatant fluid of the T cells and PBMC cells to stimulate the Hacat cells and to see if there is a proliferation of Hacat cells. We made Qingre Liangxue Jiedu drug serum, and use different concentration to intervene on the T cells (including treated by pso p27), PBMC cells (including treated by pso p27) and Hacat cells.The assays just like which were mentioned.(2)The influence of Traditional Chinese medicine monomers on the proliferation and apoptosis of Hacat cells We chose4kinds of Traditional Chinese medicine monomers:ursolic acid (Hedyotis diffusa), astilbin(Smilax glabra Roxb.), shikonin(Amebia euchroma Johnst), and Paeonol (Paeonia suffuticosa Andr). In this study, we want to know the exactly function of the monomers on Hacat cells. We tested the proliferation of Hacat cells by WST-8, and we observed the mitotic cycle and apoptosis rate of Hacat cells by flow cytometry and immunofluorescent microscope, what is more, we tested the expression of apoptosis gene Bcl-xl and Bax in Hacat cell by RT-PCR. All the cells were treated by different ratio of monomers.Result(1)Pso P27antigen cannot active T cells directly in vitro, it cannot stimulate the proliferation of T cells and have no effect on the release of correlative cytokines. But it can promote the proliferation of PBMC cells, and induce the release of TNF-a.When we add the supernatant fluid of PBMC cells it can promote the proliferation of Hacat cells. The Qingre Liangxue Jiedu drug serum can suppress the proliferation of T cells, PBMC cells and Hacat cells in vitro. The level of suppression related to the concentration of Qingre Liangxue Jiedu drug serum, when the ratio of the drug serum was added in the culture medium up to20%, it made an obviously suppression on Hacat cells, especially in the large dose group and the positive control group. At the meantime, we observed the proliferation of Hacat cells treated by drug serum for5days, but there was no clear relationship between the suppression of Hacat cells and the treated time.(2) In the research of Traditional Chinese medicine monomers and Hacat cells. We found that ursolic acid,,shikonin and paeonol can inhibit the proliferation of Hacat cells, and the inhibition related closely to the content of monomers. However, astilbin can stimulate the proliferation of Hacat cells. Moreover, c, shikonin and paeonol can block the cell cycle, but there were some differences. For example, ursolic acid can increase G1/G0phase of cell cycle, meanwhile, shikonin and paeonol can increase G2/M phase. Because astilbin can promote the proliferation of Hacat cells, so when we analyze the cell cycle invented by it, although it can decrease G2/M phase and increase G1/G0phase, we can’t regard this as cell cycle blocking. Maybe it just let more cells to go to next cell cycle. What is more, ursolic acid, shikonin and paeonol can induce the apoptosis of Hacat cells. The results showed that there was a significant statistical difference between the intervention groups and the control group in apoptosis rate. But astilbin can prevent Hacat cells from apoptosis, the significant statistical difference in early apoptosis rate between intervention groups and control group was detected too. In this reaseach, we also observed the influence of monomers on the expression of apoptosis gene of Hacat cells, such as Bcl-xl and Bax. Bcl-xl is one of apoptosis inhibiting gene and Bax is an apoptosis trigger gene, they have opposite effect on apoptosis of cells. The influence on the2genes was quite distinct among monomers. Ursolic acid can increase the expression of Bcl-x1and inhibit the expression of Bax of Hacat cells, but shikonin can inhibit the expression both of them, paeonol can increase the expression both of them. Meanwhile, Astilbin had no obvious effect on the expression of the2genes.ConclusionPso p27, is the only antigen in psoriatic lesions recognized by antibodies from psoriatic scale, to our knowledge. The mechanism of it in participating psoriasis is going to be investigated. Lots of studies have showed that T cell-mediated keratinocyte proliferation plays a key pathogenetic role in psoriasis. What is more, we thought that Pso p27can activate T cells and induce the release of cytokine, which can mediate proliferation of keratinocyte. However, the results showed there were no activation and the release of cytokines of T cells, the pso p27just activated PBMC cells. So we still don’t know the exact target cells and mechanism of pso p27made its role in psoriasis. We need some new hypothesizes and more experiments to inspect and verify.Qingre Liangxue Jiedu drug serum can inhibit the proliferation of kinds of cells related to psoriasis, and the function had a close relation to the dose of drug serum. But the drug serum had a poor stability, because the manufacture craft was complex. So we should pay attention to improving the manufacture craft. What is more, the characters of Chinese herbal compound in treating psoriasis was multi-target and multi-access, inhibition of the proliferation of cells just was one aspect of its function, we should make a all-around understanding of the Qingre Liangxue Jiedu drug serum.In this project, we also observed the function of Traditional Chinese medicine monomers on the proliferation, cell cycle, apoptosis, the expression of apoptosis genes Bcl-xl and Bax of Hacat cells in vitro. The function of these monomers had similarities, but there were still some differences. The influence on Hacat cells of ursolic acid, shikonin and paeonol was unanimous with the effect of Hedyotis diffusa, Arnebia euchroma Johnst and Paeonia suffuticosa Andrin in treating psoriasis, but astilbin can induce the proliferation of Hacat cells in vitro and prevent the apoptosis of Hacat cells, this was quite different with the effect of Smilax glabra Roxb in treating psoriasis in clinic, so we reasoned that the trigger-cell of in treating psoriasis is not karatinocye, or astilbin is not the pivotal monomer of Smilax glabra Roxb in treating psoriasis. |
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