Identification Of Screening Models For Cell Necrosis Inhibitors And Screening Of Anti-necrotic Small Molecule Compounds

programmed cell necrosis is irreversible tissue damage due to bioenergy disorders,and is a caspase-independent form of cell death.Studies have found that programmed cell necrosis has important pathophysiological significance in the occurrence and development of neurodegenerative diseases,tissue ischemia-reperfusion injury,inflammation,sepsis,multiple solid organ injury and other diseases.The inhibition or upregulation of programmed cell necrosis signal can affect the occurrence and development of some diseases,indicating that programmed cell necrosis can be a potential therapeutic target for many diseases.Further research on small molecule inhibitors of cell necrosis may be of great significance to find relevant drugs for the prevention and treatment of various diseases.In 2012,a study revealed that under oxidative stress conditions,p53 protein was able to form a complex with cyclophilin d?PPIF?in the mitochondrial matrix,opening the mitochondrial membrane permeability transition pore?PTP?,causing the release of mitochondrial body contents and thus leading to cell necrosis.In addition,the researchers detected the p53-PPIF complex in the brain tissue necrosis region of a stroke model mouse,preventing the formation of the complex helping to reduce symptoms of stroke.Mitochondrial p53-PPIF is an important contributor to oxidative stress-induced cell necrosis,inhibiting the formation of the complex may help prevent tissue damage caused by mass cell necrosis,thereby inhibiting the development of disease.In this study,we screened the inhibitors of p53 and PPIF interaction from more than 940small molecule compounds by using p53 and PPIF interaction modulator screening model.It is expected that small molecule compounds that inhibit the interaction between the p53 and PPIF can be found,with a view to further finding lead compounds that inhibit cell necrosis,and provide novel drugs for the treatment of various diseases.The research is as follows:1.Identification of p53 and PPIF interaction regulator screening model.We first co-transfected the pBIND-p53,pACT-PPIF eukaryotic expression vectors and the luciferase reporter vector pG5-luc,which have been constructed according to the principle of the mammalian two-hybrid system,into HEK293T cells using calcium phosphate transfection method.The luciferase activity results confirmed that the screening model is working well.suggested that the screening model of p53 and PPIF interaction modulator was successfully constructed and could be used in the subsequent screening experiments of small molecule compounds.2.Screening for the inhibitors of p53 and PPIF interaction from the small molecule compounds.The screening model for the modulator of p53 and PPIF interaction was used to screen more than 940 small molecule compounds stored in our laboratory.After screening,a total of 17 small molecule compounds were found to significantly inhibit the interaction between p53 and PPIF.These 17 small molecule compounds were used as candidate compounds for subsequent experiments.3.Inhibitory effects of screened out compounds on H₂O₂-induced cell necrosis.In this study,the non-toxic concentration of compounds was first detected by the MTT method.It was found that compound T4-79,T6-26,A3-49 and A3-66 had no significant effect on the viability of HK-2 cells at a working concentration of 5?g/ml.Then,we used H₂O₂ to induce HK-2 cells necrosis,and treated the cells with non-toxic concentrations of the compounds,and tested the protective effects of the compounds on HK-2 cells by MTT method.The results showed that compounds T4-79,T6-26,A3-49 and A3-66 could significantly increase the cell viability of HK-2 cells induced by hydrogen peroxide.Trypan blue staining results also showed that the pretreatment with these four compounds can significantly reduce the positive staining cell rate with trypan blue,indicating that these four target compounds could inhibit H₂O₂-induced cell necrosis.To further verify this result,we performed PI staining experiment.The results showed that pretreatment with these four screened out compounds could significantly reduce the H₂O₂-induced positive rate of PI staining.We then tested the lactate dehydrogenase?LDH?activity in the cell culture supernatant.It was found that pretreatment with the target compounds can significantly reduce the H₂O₂-induced LDH release compared with the DMSO+H₂O₂ treatment group,indicating that pretreatment with the target compounds can alleviate the H₂O₂-induced oxidative damage of HK-2 cells.Finally,the results of Annexin-V-FITC and PI double staining flow cytometry showed that the PI positive staining rate in the H₂O₂ treatment group was significantly increased when compare with the DMSO control group,and T4-79,T6-26 target compound pretreatment can significantly decrease the PI positive staining rate when compared with the H₂O₂ treatment group,indicating that T4-79 and T6-26 could inhibit H₂O₂-induced HK-2 cells necrosis.To further explore the mechanism of the cell death,we used Western blot method to detect the levels of MLKL and p-MLKL in HK-2 cells.The results showed that the p-MLKL protein level was significantly higher in the H₂O₂ induced group compared to the DMSO-treated group,however the p-MLKL protein level was down-regulated after pretreatment with the T4-79,A3-49,T6-26 or A3-66 target compounds.The above results suggested that all four compounds can inhibit programmed necrosis of cells.In summary,in this study,by using the PPIF and p53 interaction regulators screening models,four small molecule compounds T4-79,A3-49,T6-26,A3-66 that can inhibit H₂O₂-induced HK-2 Cell necrosis were screened out,that provides a new ideas for the further development of leading compounds of cell necrosis treatment drugs,and lays a material foundation for the treatment of various diseases such as stroke and ischemia-reperfusion injury.