Genetically Encoded Reactive Oxygen Species Indicators Based On Keap1/Nrf2-ARE Signaling Pathway

When the body is exposed to harmful stimuli,Intracellular oxidative stress reactions appear,and reactive oxygen species(ROS)in the organism is one of the indicators in the reactions.When the free radicals are generated excessively,the degree of oxidation exceeds the scavenging capacity of oxides.Oxidative and antioxidant systems can become unbalanced,leading to tissue damage[1].In order to detect and capture the body’s ROS levels above normal physiological levels in living cells,Keap1(Epoxy Chloropropane Kelch Sample Related Protein-1,Kelch-like epichlorohydrin-related protein-1)/Nrf2(Nuclear Factor Erythroid-2 related Factor 2,Nuclear Factor E2 Related Factor 2)-ARE(Antioxidant Response Element)endogenous antioxidant signaling pathway[2]was investigated.It can be modified as an endogenous detection new way of ROS.The Keap1/Nrf2-ARE signaling pathway is a very important pathway in the organism following oxidation-antioxidants systems,that has not yet been studied and modified,and the fluorescent proteins have a simple transformability and many advantages in live cell imaging.Therefore,this topic aims to complete the ROS detection fluorescence bio-cell sensor coded at the gene level based on Keapl/Nrf2-ARE signaling pathway,applied to the detection of ROS damage caused by environmental toxicants and oxidative damage to tumor cells.This study designed and constructed a successful endogenous ROS cell sensor based on the Keapl/Nrf2-ARE signaling pathway,and tested its performance and application.Firstly,the ROS cell sensor was constructed by constructing a plasmid expression vector for each element,mCherny and mNeonGreen fluorescent protein were selected as response signals to construct cell sensors,and the best choice was based on the specific detection items.Secondly,the cell sensor was tested by H2O2 exogenous stimulation.At the same time,the sensor optimization work was carried out and reassessed.It was found that mNeonGreen has superior sensitivity under the exogenous stimulation of H2O2.Finally,we studied the actual application of the ROS cell sensor test work,on the one hand,select the use of paclitaxel and resveratrol,which be drugs increase organism’s ROS level for detection experiments,found that low concentrations of paclitaxel will increase the ROS level of cells,the fluorescence signal increases follows;the concentration of resveratrol is directly proportional to the sensitivity of the fluorescence signal,and the high concentration of resveratrol causes a greater increase in ROS and a more pronounced fluorescence signal.On the other hand,the constructed ROS cell sensor was transferred into leukemia cell HL-60 for detection of oxidative damage.Experiments showed that the physiological ROS level of tumor cells was higher than normal human cells.Under the exogenous stimulation with high concentration of H2O2,the cell sensor fluorescence signal appears more pronounced.The future development prospects of this genecically encoded ROS indicators are more extensive.For example,from basis of this study,quantitative analysis of exogenous stimulation and ROS can be performed by quantifying fluorescence signals,which can be associated with elevated levels of ROS;and it can be applied to other tumors cells’detection;it’s worth mentioning that the Keapl/Nrf2-ARE signaling pathway has great significance in the oxidation and antioxidant system in human,but many mechanisms are still unknown,and this reaserch also provides some basic studies for the in-depth study.