Regulation Mechanism Of NRF2 On Cell Clock Gene Oscillation In Simulated Microgravity Environment

Background: Circadian is an evolutionary conservative autonomous timing mechanism that plays an important role in regulating body behavior and physiology.It coordinates biological behavior patterns that are consistent with the environment,and regulates physiological functions by predicting environmental changes.The circadian rhythm timing mechanism is based on a transcription/translation feedback loop,which is mainly regulated by the interaction of four proteins: CLOCK,BMAL1,Period(PER)and Cryptochrome(CRY).CLOCK and BMAL1 can form heterodimers and bind to the E-box upstream of Per and Cry to activate their transcription,causing PER and CRY to accumulate in the cytoplasm and form heterodimers,which enter the nucleus in a negative feedback loop.Negatively regulate the transcriptional activation of Clock and Bmal1.The state of microgravity refers to the gravity of an object is at the nanometer level.The space flight environment is a microgravity environment.In order to ensure the normal progress of space operations,a lot of pre-research on the ground is required.Microgravity simulation technology has been widely used as the main research method.A number of space flight and ground simulation microgravity experiments have shown that exposure to a simulated microgravity environment can cause sleep disorders,heart rate and body temperature rhythm changes,indicating that microgravity significantly affects the biological circadian rhythm.Nuclear factor erythroid-2 related factor 2,Nrf2,as an antioxidant transcription factor,is regulated by oxidative signals and enters the nucleus and combines with the Anti-oxidative response element(ARE)in the promoter region of downstream genes to promote its transcription.It has been reported in the literature that simulated microgravity can significantly increase the level of reactive oxygen species(ROS)in the brain tissue of rats,and the increase in cellular ROS levels can activate NRF2 activity.In addition,studies have shown that NRF2 can indirectly inhibit the transcription of rhythm genes mediated by CLOCK-BMAL1 by regulating the expression of Cry2 and Rev-erb,and transmit cellular redox signals to the circadian rhythm.We use the transcription factor binding site prediction database JASPAR to find that there are ARE elements in the promoter regions of the rhythm genes Clock and Bmal1 that can bind to NRF2.For this reason,we speculate that NRF2 may act by directly binding the ARE elements in the Clock and Bmal1 promoter regions,NRF2-ARE pathway may be an important way to regulate clock gene oscillation in microgravity environment.Objectives:(1)To reveal the effect of simulated microgravity on the expression of Nrf2.(2)To investigate the role of NRF2-ARE pathway in regulating the expression of clock genes in simulated microgravity environment.Methods:(1)The clinostat simulates the microgravity effect.Horse serum synchronize with the NIH/3T3 cells.After continuous clinorotation in the clinostat for 24 hours,samples are collected every 4 hours for 24 hours.Western blotting is used to detect the expression of Clock,Bmal1 and Nrf2.DCFH-DA fluorescent labeling to detect ROS level,and nuclear separation test to detect NRF2 entering the nucleus;(2)Knock down Nrf2 or treat NIH/3T3 cells with the NRF2 degrading agent Brusatol.Horse serum induces cell synchronization.Samples are collected every 4 hours for 24 hours.Western blotting was performed to detect the expression levels of Clock,Bmal1 and Nrf2,and verify the influence of NRF2 on the expression rhythm of the rhythm genes Clock and Bmal1;(3)Use lipofectamine 3000 to transfect NIH/3T3 with a plasmid containing the ARE element in the Clock promoter region,a plasmid containing a mutant ARE element in the Clock promoter region,and an empty plasmid for 24 hours.The dual luciferase reporter gene assay detects the binding ability of NRF2 and the ARE element in the Clock promoter region;(4)Use lipofectamine to transfect NIH/3T3 cells with Nrf2 overexpression plasmid for 24 h.Synchronize with horse serum for 2h.After continuous clinorotation for 24 h in the clinostat,samples are collected every 4h,Western blotting was performed to detect the expression levels of Clock,Bmal1 and Nrf2 to verify the effect of Nrf2 on rhythmic recovery;(5)NIH/3T3 cells were treated with 0?M,10?M,20?M,40?M,80?M,160?M,320?M,640?M and 1280?M luteolin for 24 h,and Western blotting was used to detect the expression levels of Clock,Bmal1 and Nrf2;20?M luteolin treatment NIH/3T3 cells for 24 h,clinorotation for 24 hours,the expression levels of CLOCK,BMAL1 and NRF2 were detected by Western blot experiments to explore the effect of luteolin on the expression rhythm of CLOCK and BMAL1 by reversing the simulated microgravity effect by promoting the expression of NRF2.Results:(1)The clock genes Clock and Bmal1 were expressed rhythmically under normal conditions;The simulated microgravity effect weakened the oscillation rhythm of Clock and Bmal1,shortened the period and changed the phase.(2)The simulated microgravity effect increased the level of cellular ROS(P <0.05),the expression of rhythm genes Clock and Bmal1 decreased(P <0.05),the expression of antioxidant transcription factor NRF2 decreased(P <0.05),and the nucleus entry decreased;(3)Horse serum can induce the rhythm of Clock and Bmal1.NRF2 is an essential factor involved in the rhythmic expression of Clock and Bmal1;NRF2 can promote the expression of Clock and Bmal1(P <0.05);(4)There is an ARE element that binds to NRF2 in the Clock promoter region;NRF2can directly bind to the Clock-ARE element to regulate the expression of Clock;the simulated microgravity effect reduces the binding ability of NRF2 and Clock-ARE(P<0.05);(5)Overexpression of NRF2 can reverse the rhythmic changes caused by the microgravity effect and enhance the rhythmicity of Clock and Bmal1;(6)Luteolin can promote the expression of Nrf2 and reverse the decrease of Nrf2 expression caused by the simulated microgravity effect.Conclusions:(1)Simulated microgravity effect can lead to decreased levels of Nrf2 protein,increased levels of ROS,and decreased expressions of Clock and BMAL1.(2)Simulated microgravity effect can directly regulate the oscillating rhythm of circadian gene Clock through the Nrf2-ARE pathway.The Nrf2-ARE pathway may become an important target for circadian rhythm regulation in microgravity environment...