Effect Of PF On Proliferation, STAT-3 Activation And MMP-2, VEGF Secretion In HaCaT Cell Induced By TNF-a

Paeoniflorin which is extracted from the root of peony plants has wide pharmacological effects such as anti-inflammatory, immune-adjustability, kidney and liver protection, anti-depressants and influencing cell proliferation, accounting for 90% of TGP. It is found that TGP possesses the true effect on treatment of psoriasis in clinical study.Psoriasis, a chronic inflammatory and proliferative dermatosis, is a stubborn disease, and offen occurs in young, which posses the character of recurrence and progressive increase and thus it has done a great harm to the human body. The pathology of psoriasis presents excessive proliferation and differentiation of keratinocyte, infiltration of abnormal inflammatory cell and extension, relocation and expansion of dermal capillary proliferation. STAT-3, involved in intracellular signal transduction and regulation of the cytokine network, plays an important role in regulation of gene expression of many cytokines, inflammatory mediators and regulation of immune responses and is involved in the pathogenesis of psoriasis as well. Therefore, STAT-3 is an important target for the treatment of psoriasis.The purpose of dissertation is to illustrate the mechanisms of paeoniflorin from the new perspective points of STAT-3 and to provide basic principle and experimental evidence for the clinical application of paeoniflorin in the treatment of psoriasis and proliferative skin disease, through the investigation of the effect of paeoniflorin on STAT-3 activation and STAT-3 activation on inducing cell proliferation, downstream cytokine MMP-2 and VEGF secretion.①TNF-a induce the proliferation of HaCaT cell, its STAT-3 activation and the secretion of MMP-2 and VEGFHaCaT cells are induced by 10ng/ml TNF-a and then morphological changes of HaCaT cells are observed using inverted microscope. The results indicate that the cell numbers in the stimulation group significantly increase but cell morphology has no significant difference, compared with normal contrast group. And also, it is found that cell proliferate significantly up to 201% after TNF-a stimulation by means of MTT colorimetric assay of cell proliferation rate. Whereas, by western-blot detection of activated STAT-3 (p-STAT-3), it is found that p-STAT-3 expression does not exist without stimulation, but the expression of p-STAT-3 goes up significantly after TNF-a stimulation. In addition, through detection of STAT-3mRNA and its downstream cytokine MMP-2 and VEGF mRNA expression activated by STAT-3 using fluorescence quantitative PCR, the result indicates that the expression of STAT-3mRNA does not change significantly after stimulation while MMP-2 and VEGF expression increase significantly.②Effects of paeoniflorin on HaCaT cell proliferation, STAT-3 activation and MMP-2, VEGF secretion induced by TNF-aThe experimental group are marked as the time gradient group and the concentration gradient of group respectively and then morphological changes of HaCaT cells in concentration and time groups are observed with inverted microscopy. The results indicate that cell morphology in various concentrations and time group are normal and there are no significant differences among the various concentration groups. However, compared with TNF-a stimulation group, cell densities are a little sparser, exceptionally significant after 24h intervention. Assaying inhibition of HaCaT cell proliferation after TNF-a stimulation under the intervention of paeoniflorin using MTT colorimetric assay, it is found that proliferation inhibition rate of HaCaT cells induced is time and concentration dependent. In addition, changes of the activation of STAT-3 (p-STAT-3) in various concentrations using western-blot detection method indicate that concentration has no significant reverse effect on the activation of STAT-3. At last, using fluorescence quantitative PCR to detect the changes of STAT-3mRNA and of the mRNA of downstream cytokine MMP-2, VEGF expression in the intervention group, it is found that intervention has no significant effect on the expression of STAT-3mRNA, but the expression of MMP-2 and VEGFmRNA in HaCaT cells has a certain degree of reduction.To sum up, experiment results indicate that paeoniflorin has the ability to inhibit the proliferation of HaCaT cell induced by TNF-a and to reduce the expression of cytokine VEGF and MMP-2 in psoriasis-related. However, it has no significant reversed effect on activated STAT-3.