Construction Of Screening Model For Necrosis Inhibitors And Screening The Inhibitors From Traditional Chinese Medicine

Necrosis is a common phenomenon in the cardiovascular and cerebrovascular diseases,neurodegenrative diseases and so on.Recent investigation showed that p53 accumulated in the mitochondrial matrix,triggered mitochondrial permeability transition pore(PTP)opening and cell necrosis by physical interaction with the PTP regulator cyclophilin D(CypD).If we can effectively intervene the process of pathological cell necrosis,may prevent the secondary tissue injury caused by necrosis and improve patients’ symptoms.In this paper,we targeted that p53 opened the mitochondrial permeability transition pore to trigger necrosis and constructed the interaction model of p53 and CypD,then we screened the inhibitors of the p53-CypD interaction from Chinese medicine by using this model,in order to find the candidate compounds which can inhibit necrosis.Our work can provide experimental basis for development of necrosis inhibitor.1.Construction of CypD gene eukaryotic expression vector pcDNA3-CypD.The total RNA was extracted from L02 cells,then the CypD coding sequence was amplified by RT-PCR and inserted into eukaryotic expression vector pcDNA3.We confirmed the CypD coding sequence we identified was consistent with the CypD sequence in GenBank.At last,we transiently tranfected pcDNA3-CypD into HEK293T cells and tested the expression of CypD by western blot,the results showed that we have successfully constructed the CypD gene eukaryotic expression vector.2.Construction of the screening model for identifying p53-CypD interaction regulator.According to the principle of mammalian two-hybrid system,we first constructed of the eukaryotic expression vector pACT-p53 and pBIND-CypD respectively.Afterwards we transfected pACT-p53,pBIND-CypD and the reporter vector pG51uc into HEK293T cells simultaneously,then tested luciferase activity,the result indicated that this screening model was effective and can be used in subsequent screening experiments.3.Screening for the inhibitors of p53-CypD interaction.We screened more than 300 compounds extracted from traditional Chinese medicine by using this screening model and 2 compounds,named TI45 and TI89 were identified to show inhibitory effect on the interaction of p53 and CypD.4.Validation of protective effect of candidate compounds on cell necrosis.We induced HEK293T cells necrosis with H2O2 and confirmed the protective effect of candidate compounds on necrosis through MTT assay and trypan blue stain experimentThis study provided a new method for screening cell necrosis inhibitor and laid the material fundation for development of cell necrosis inhibitor.