Drug Screening Model Construction Based On Inhibiting NF-κB Signal Pathway

Nuclear factor kappaB (NF-κB), which widely existed in eukaryotic cell, was first identified over 20 years ago as a DNA-binding complex governing transcription at the immunoglobulin light chain gene intronic enhancer in mature plasmocytes. Via modulating the expression of related target genes, NF-κB plays a critical role in diverse cellular processes associated with proliferation, cell death, development, as well as innate and adaptive immune responses, especially in inflammation and tumorgenesis, and is considered as an ideal target for the prevention and treatment of cancer and inflammation. So, it is extremely important to set up a drug screening model based on inhibiting NF-κB signal pathway.As the development of biomedical sciences and biotechnologies, the high throughput drug screening was set up in the end of the 20th century, which based on the random selection. It can be used for screening a large scale of samples and become an important technique for the drug searching initiatively. High throughput screening technique can be used in the yeast-based platform, cell-based platform and animal-based platform by the means of molecular and cellular experimental methods. Luciferase reporter gene technique plays an important role in high throughput screening.It is demonstrated that TNFa can be used as a positive stimulator for NF-κB signal pathway, because TNFa can activate NF-κB properly both in vivo and vitro. IκBαcombines with NF-κB in the cytoplasm, which prevents NF-κB locating into the nucleus and executing its transcriptional activity, so we can presume the activity of NF-κB through detecting the degradation level of IκBα. Gambogic acid (GA for abbreviation) can be used as a positive inhibitor for NF-κB signal pathway, because GA can inhibit the activity of NF-κB. In this article, we find out the effects of processing concentration and time of TNFa on the expression of NF-κB-luc reporter gene and the degradation level of NF-κB inhibitory subunit IκBα, using luciferase assay and western blot assay respectively. Therefore, we can construct a drug screening model based on inhibiting NF-κB signal pathway.In the luciferase reporter gene screening model, we tested the most proper processing concentration and time of TNFa on 293T cells. The results showed, the expression level of NF-κB reporter gene was highly enough by the stimulation of 0.01 nM TNFa in 293T cells for 24 h, which directly related to the concentrations and processing time of TNFα. The screening model is confirmed by the known NF-κB positive inhibitor gambogic acid.In the NF-κB inhibitory subunit screening model, we tested the most proper processing concentration and time of TNFαon Panc-28 cells. The results showed, IκBαdegraded notably at the density of 0.01 nM after 5 min treatment with TNFαon the Panc-28 cancer cells, and almost completely degraded after 20～30 min. The screening model is confirmed by the known NF-κB positive inhibitor gambogic acid.Taken together, both luciferase reporter gene screening model and NF-κB inhibitory subunit screening model could be used to select the potential NF-κB signal pathway inhibitory drugs.