The Mechanism Research On HaCat Cell Regulated By The Therapeutic Principle Of Heat-clearing, Blood-promoting And Toxicity-releasing Through PI3K/Akt Signaling Pathway

Background:Psoriasis is a common, chronic and recurrent disease, which has the genetic background and is affected by many factors such as environment, infection etc. at the same time. Many scholars consider psoriasis is an autoimmune systemic inflammatoryskin disease dominanted by Thl-Th17 axsis, complicated with cardiovascular diseases, diabetes mellitus, metabolism syndrome, depression and inflammatory bowel disease and so on. There are more and more researches about the immunologic mechanism of psoriasis, focusing on the interreaction between immunocyte, genetic regulation, and inflammatory molecules. And the abnormal of signaling pathways may play a pivotal role.Objective:To study the mechanism on Hacat cell regulated by the therapeutic principle of Heat-clearing, Blood-promoting and Toxicity-releasing (compounds and traditional Chinese monomer) through the signaling pathway, and to research the mechanism about psoriasis vulgaris treated by the therapeutic principle of Heat-clearing, Blood-promoting and Toxicity-releasing and to determine the approach targets.Methods:first of all, the HaCat cell induced by TNF-a in 24h, and then blocked by LY294002, a specific blocker on PI3K/Akt signaling pathway, in 1h. To observe the biological behaviour of TNF-a induced HaCat cell impacted by the Heat-clearing, Blood-promoting and Toxicity-releasing compounds and astilbin, and the regulation effect about PI3K/Akt signaling pathway. The proliferation was tested by CCK-8; the expression of Akt and CREB about the pathway, and apoptosis genes bax, bcl-xl and c-fos were tested by Western blotting and RT-PCR; the cell cycle and apoptosis were assayed by flow cytometry; the secretion of cytokines IFN-y, IL-1β, IL-6 and IL-8 were assayed by enzyme-linked immunoadsordent assay (ELISA).Results:(1) the Heat-clearing, Blood-promoting and Toxicity-releasing compounds could inhibit the expression of Akt on PI3K/Akt signaling pathway, and there was dose dependent on Akt protein. After the pathway blocked, the inhibition still existed, but no statistical significance compared with blocking before. At the same time, the compounds could inhibit the expression of CREB, especially in the high dose (30mg/ml). After the pathway blocked, the inhibition weakened.The Heat-clearing, Blood-promoting and Toxicity-releasing compounds could inhibit the proliferation of TNF-α induced HaCat cell. After the pathway blocked, the proliferation continued to be inhibited.The Heat-clearing, Blood-promoting and Toxicity-releasing compounds could promote the early apoptosis of TNF-α induced HaCat cell. After the pathway blocked, the early apoptosis rate continued rise, and the late apoptosis rate descended.The expression of bax was up-regulation by the Heat-clearing, Blood-promoting and Toxicity-releasing compounds, and there was dose dependent on the up-regulation. After the pathway blocked, the up-regulation weakened, especially in the high dose. The compounds could inhibit the expression of bcl-xl, and the inhibition continued to be enhanced after the pathway blocked. The compounds also could inhibit the expression of c-fos in TNF-α induced HaCat cell, and there was no significant change after the pathway blocked.The Heat-clearing, Blood-promoting and Toxicity-releasing compounds could inhibit plenty of cells in S phase to inhibit the proliferation of HaCat cell. After the pathway blocked, cells in G0/G1 phase increased and decreased in S phase.The Heat-clearing, Blood-promoting and Toxicity-releasing compounds could inhibit the secretion of IFN-γ, IL-1β, IL-6 and IL-8. PI3K/Akt signaling pathway played an important role in the regulation on IFN-γ, IL-1βand IL-6 treated by the compounds, but there was no relativity between the IL-8 and this pathway.(2) Astilbin could inhibit the expression of Akt in TNF-αinduced HaCat cell, and there was dose dependent on Akt protein. After the pathway blocked, the inhibition weakened. Astilbin could inhibit the expression of CREB, and there was dose dependent on CREB protein as well. The inhibition continued to strengthen after the pathway blocked.Astilbin could inhibit the proliferation of TNF-α induced HaCat cell. After the pathway blocked, the inhibition weakened than before. But as time went on, the inhibition strengthened compared with the pathway unblocked.The expression of bax was up-regulation by Astilbin, and increased with dose. After the pathway blocked, the up-regulation strengthened. Astilbin could inhibit the expression of bcl-xl, and the up-regulation strengthened after the pathway blocked. Astilbin could inhibit the expression of c-fos, but the inhibition weakened after the pathway blocked.Astilbin could increase the number of cells in S phase in TNF-a induced HaCat cell, decreased the number in G0/G1 phase and G2/M phase. After the pathway blocked, the number of cells in G0/G1 phase and G2/M phase continued decrease, and increased in S phase.Astilbin could inhibit the secretion of IFN-y, IL-1β, IL-6 and IL-8, and there were dose dependent on the secretion of IFN-y, IL-1β, IL-6. PI3K/Akt signaling pathway played a role in the secretion of IL-1β, but associated obviously with the secretion of IFN-γ, IL-6 and IL-8. Conclusion:PI3K/Akt signaling pathway played an important role in the proliferation, apoptosis and the secretion of cytokines in HaCat cell. The intervention of the Heat-clearing, Blood-promoting and Toxicity-releasing compounds in HaCat cell was associated with the PI3K/Akt signaling pathway. Astilbin could inhibit the proliferation of TNF-a induced HaCat cell. The intervention of biological behavior on TNF-α induced HaCat cell treated by Astilbin might be not mainly through PI3K/Akt signaling pathway, more probably via the regulation on CREB.