Thalidomide Keratinocyte Cells Secrete Vegf And Tnf-¦Á

BackgroundThalidomide is a sedative drug introduced in the late1950s that was used to treat morning sickness. It was sold from1957until1961. Although thalidomide was withdrawn after being found to be a cause of birth defection in the1960s, recently, thalidomide was approved to be used to treat erythema nodosum leprosum and multiple myeloma by the FDA due to its effect that reducing the level of tumor necrosis factor-a (TNF-a) and antiangiogenie activity. Since then, it has also emerged as a useful treatment option for many refractory dermatologic disorders such as aphthous stomatitis, Behcet's syndrome, lupus erythematosus, prurigo nodularis, and adult langerhans cell histiocytosis. But the mechanisms of the thalidomide in the treatment of dermatologic disorders are still uncertain. Moreover, because of the immunomodulatory activity of thalidomide, scientists believed that thalidomide may be able to play a role in the treatment of psoriasis. So in the previous clinical practice, we performed a pilot study to investigate the efficacy and safety of thalidomide in the treatment of psoriasis, and the results proved that thalidomide was effective for some patients. Prior studies have suggested dermal dendritic cells, T lymphocytes, keratinocytes, together with their inflammatory cytokines, such as IL-12, IL-23, IL17, IL-22, TNF-a, and VEGF, play critical roles in the pathogenesis of psoriasis. The effect of thalidomide on dendritic cells and T lymphocytes has been extensively investigated, however, there are quite a few studies in the literature regarding its effect on keratinocytes. So the mechanisms of thandomide needed to be further investigated.ObjectivesThe hypothesis of a cytokine network in psoriasis proposed a central role of pro inflammatory cytokines, including TNF-a. Acanthosis caused by keratinocyte proliferation and exaggerated vascularity are the key histologieal features of a psoriatic plaque. These theories have been validated by the tremendous clinical success of anti-TNF therapy and methotrexate in the treatment of psoriasis. Also, the efficacy of drugs targeting angiogenesis in animal models of psoriasis confirmed the role of VEGF in the psoriasis. So, the aim of our study was to investigate the mechanism of the thalidomide in the treatment of dermatologic disorders and to provide the laboratory basis for the use of thalidomide in the treatment of psoriasis by studying the effects of thalidomide on the keratinocytes viability and on the expression of VEGF and TNF-a of the human keratinocytes.Methods1. HaCaT cells were cultured in vitro.2. Cell viability affected by different concentrations of thalidomide was measured by WST-1assay.3. The2-△△Ct method was validated by regulating the PCR efficiency.4. Quantitative real-time PCR was performed to determine the mRNA levels of VEGF and TNF-a in the HaCaT cells that incubated with thalidomide on different time points.5. Quantitative real-time PCR was performed to determine the mRNA levels of VEGF and TNF-a in the HaCaT cells that incubated with different concentrations of thalidomide.6. Sandwich ELISA was used to detect the protein levels of VEGF and TNF-a in the HaCaT cells that incubated with different concentrations of thalidomide.Results1. Cell viability of HaCaT cells was not impacted by the thalidomide when the concentration of thalidomide was not higher than100nM. At1000nM thalidomide, the percentage of viable cells was reduced to90.8%compared with the control group. The cell viability was decreased by the thalidomide in the concentration range of1000nM to100000nM in a concentration-dependent manner.2. The relative expression level of VEGF and TNF-a mRNA was significantly suppressed at3hours when the relative expression level of VEGF and TNF-a mRNA decreased to51.0%and53.1%respectively than that of other time points.3. The relative expression levels of VEGF mRNA and protein were reduced to63.4%and92.3%respectively by the thalidomide at the concentration of0.01nM. The relative expression levels of VEGF mRNA and protein were attenuated by the thalidomide in the concentration range of0.01nM to100nM in a concentration-dependent manner.4. Thalidomide that at the concentration not more than0.01nM did not affect the expression of the mRNA and protein of TNF-a. The relative expression level of TNF-a mRNA and protein were reduced to58.7%and61.7%respectively by the thalidomide at the concentration of0.1nM. TNF-a mRNA expression level was down-regulated by thalidomide when the concentration of thalidomide in the range of O.1nM to100nM, and TNF-a protein expression levels were decreased by thalidomide when the concentration of thalidomide in the range of0.1nM to100nM in a concentration-dependent manner.Conclusions1. Thalidomide reveals cytotoxicity when its concentration is not lower than1000nM.2. The expression of mRNA and protein of VEGF and TNF-a can be suppressed by thalidomide.3. Thalidomide cloud be useful in the treatment of psoriasis due to the activity of reducing the expression of VEGF and TNF-a. Further investigation should be pursued.