Effect Of Total Glucosides Of Paeony On Cell-Proliferation And Expression Of Vascular Endothelial Growth Factor And IL-23 In Human HaCaT Keratinocytes

BackgroundPsoriasis is a common, chronic, inflammatory disease. The cause and pathogenesis of psoriasis is still unknown. The disease is characterized by keratinocyte hyperproliferation, vascular hyperplasia and infiltration of T lymphocytes, neutrophils, and other types of leucocytes in affected skin. It is generally believed to be a complex autoimmune inflammatory disease with a genetic basis. Cytokine production by keratinocytes has multiple consequences for the migration of inflammatory cells,may have systemic effects on the immune system,influences keratinocyte proliferation and differentiation processes,and finally affects the production of other cytokines by keratinocytes.VEGF is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth and chronic inflammation. VEGF is expressed and secreted by epidermal keratinocytes in normal human skin. Keratinocytes overexpress VEGF in clinically involved and uninvolved skin of patients with chronic plaque psoriasis. In transgenic mice with epidermis-specific overexpression of VEGF and enhanced skin vascularity and vascular permeability, chronic transgenic delivery of VEGF to the skin induced inflammation and all characteristics of psoriasis spontaneously, and the VEGF antagonist reversed the phenotype. These findings indicate VEGF might play an important role in the pathogenesis of psoriasis. IL-23 is produced by dendritic cells, other antigen-presenting cells and keracinocytes. IL-23 is clearly elevated in psoriasis lesions.Importantly, IL-23 levels decrease with clinical improvement of psoriasis following effective treatment, providing a direct correlation between overproduction of IL-23 and active psoriasis.In other mouse studies, recombinant IL-23 injected into normal-appearing skin produced erythematous,thick, scaly skin, with histologic features reminiscent of psoriasis.Paeonia lactiflora Pall is a kind of Chinese traditional herbal medicine. Total glucosides of paeony (TGP) consists of more than 90% paeoniflorin and other components such as hydroxypaeoniflorin, paeonin, albiflorin,benzoylpaeoniflorin,etc. Previous studies have demonstrated that TGP exerts anti-inflammatory,analgesic effects in the rat model of AA and carrageenan-induced arthritis. However, the mechanism of TGP on psoriasis remains unclear and need further investigation.The mitogen-activated protein kinase (MAPK) pathways are the best characteriz-ed of intracellular protein kinase cascades. MAPK plays a role in signal transduction associated with cell proliferation, differentiation and the production of cytokines. There are three well-characterized MAPK subfamilies in mammalian cells:extra cellular-signal-regulated protein kinase 1/2 (ERK1/2), the p38 mitogen-activated protein kinases (p38 MAPK) and the c-Jun N-terminal kinase (JNK). A strong link has been established between the p38 pathway and inflammation.The activation of the p38 pathway plays essential roles in the production of proinflammatory cytokines TNF-aand IL-1.However, little is known about the exact role of TGP in HaCaT cells. We hypothesized that TGP may modulate VEGF and IL-23 expression. We examined whether this effect functioned via the mitogen-activated protein kinases (MAPKs) signal transduction pathway, particularly p38 mitogen-activated protein kinase (p38 MAPK). In light of this, we performed a study to investigate the effect of total glucosides of paeony(TGP) on cell-proliferation and secretion of inflammatory cytokines VEGF and IL-23 in human HaCaT keratinocytes and whether p38 pathway is involved in this progress.Objective1. Investigate the effect of TGP on the proliferation of HaCaT cells;2.Investigate the effect of CRH on the expression of VEGF and IL-23 on HaCaT cells;3. Examine whether the effect of TGP on the expression of VEGF and IL-23 functioned via MAPKs signal transduction pathway.Materials and methods1. Cell cultureThe immortalized human HaCaT keratinocytes were maintained at 37℃and 5% carbon dioxide (CO2) in 1640 supplemented with 10% heat-inactivated fetal bovine serum,100 U/ml penicillin and 100μg/ml streptomycin.2. Cell pretreatmentHaCaT cells were seeded at density 4×104 cells/ml, grown for 48 h until 70% confluence. The cells were pretreated with 10μM SB203580 and incubated for 2 h before application of TGP.3. Methods3.1. MTT assay for cell proliferationHaCaT cells were incubated with TGPof different concentrations respectively, and then followed by MTT assay.3.2 Real-time RT-PCRAfter the experimental treatment, the total RNA was extracted from HaCaT cells. The reverse transcription of RNA to cDNA was performed. cDNA were amplified with real-time RT-PCR. VEGF mRNA and IL-23 mRNA expression were normalized to the expressed housekeeping gene human GAPDH. Samples were tested in triplicate and the average values were used for quantification.3.3. ELISAAfter stimulation for 48 h, culture supernatants of cells were collected, centrifuged (15 000 rpm,5 min) and stored at -80℃until analysis. The concentrations of VEGF and IL-23 in the culture supernatant were measured by commercially available enzyme linked immunosorbent assay (ELISA) kits according to manufacturer's instructions. Each supernatant was analyzed in triplicate.3.4. Western blotThe phosphorylation of p38 MAPK was detected by western blot analysis, SB203580 was used to block p38MAPKs pathway. Densitometric analysis of the band intensity was carried out using Qwin software.Results1. TGP inhibited HaCaT proliferationTGP increased HaCaT proliferation in a lower concentration and TGP 12.5mg/L～125mg/L inhibited keratinocytes proliferation distinctly.2. Effect of TGP on VEGF production in HaCaT cellsTGP increased the expression of VEGF mRNA and protein in a lower concentration and TGP inhibited the expression of VEGF mRNA and protein in a higher concentration.3. Effect of TGP on IL-23 production in HaCaT cellsTGP increased the expression of IL-23 mRNA and protein in a lower concentration and TGP inhibited the expression of IL-23 mRNA and protein in a higher concentration4. The role of MAPKs pathway in the effect of TGP in HaCaT cells 4.1 TGP activated p38 MAPK phosphorylation in HaCaT cellsTGP induced a rapid phosphorylation of p38 MAPK in a time dependent manner and with a peak at 5 min.4.2. The effect of p38 MAPK inhibitor SB203580 on phosphorylation of p38 MAPK in HaCaT cellsPretreating HaCaT cells with the p38 MAPK inhibitor SB203580 inhibited the TGP-induced phosphorylation of p38 MAPK. These data indicated that TGP activated p38 MAPK phosphorylation in HaCaT cells.Conclusions1. TGP inhibited keratinocytes proliferation;2. VEGF and IL-23 mRNA expression and protein production were downregulated by TGP;3. TGP induced a rapid phosphorylation of p38 MAPK4. TGP downregulated VEGF,IL-23 expression in HaCaT cells by p38 MAPK pathways.