Protective Effect And Mechanism Of TAZ On Microglia Cells In Inflammatory Response

TAZ is the core protein of the Hippo signaling pathway.When the Hippo signaling pathway were inhibited,TAZ are dephosphorylated and moved to the nucleus,then binds to the TEAD and regulated gene expression.Changes in TAZ expression are accompanied by changes in reactive oxygen species(ROS).Keap1-Nrf2 signal pathway can maintain the stability of intracellular ROS.When Nrf2 are activated,the expression of Nrf2 downstream protein like SOD,CAT and GR and other antioxidant enzymes will be increased,thereby reducing intracellular ROS level.ROS also as a mediator of cell inflammation can mediate the neuroinflammation occurrence.Neuroinflammation can exacerbate the development of neurodegenerative diseases,and microglia mediate the development of neuroinflammation.Inhibiting the excessive activation of microglia can effectively relieve neurodegenerative diseases.In this study,BV2 cells were cultured in vitro and construct a TAZ overexpression vector.We researched the effect and mechanism of TAZ on BV2 cells in inflammatory response.First,the effect of TAZ on LPS-induced microglia inflammation was studied.The expression of TAZ in BV2 cells after LPS stimulation was detected by qRT-PCR and Western blot.After overexpression of TAZ,LPS-induced inflammation model was used for subsequent experiments.The secretion of TNF-?,IL-6 and IL-1? was detected by ELISA,and the mRNA expression of TNF-?,IL-6 and IL-1? was detected by qRT-PCR.The results show that overexpression of TAZ can inhibit the expression of three pro-inflammatory factors.Severe oxidative stress is often accompanied by cellular inflammation,so we use corresponding kits to detect changes in SOD,GPX,GR and CAT activity and the ratio of antioxidants GSH to GSSG;MDA test kit to determine the content of lipid oxidation products MDA Changes;total cellular ROS and O2-were detected by flow cytometry.The results showed that TAZ can reduce the level of oxidative stress in cells.ROS are mainly produced by mitochondria.Excessive ROS can cause the disorder of cellular mitochondria function.In order to explore the protective effect of TAZ on mitochondria,mtROS and mitochondrial membrane potential were detected by flow cytometry;ATP content was determined by ATP detection kit.The results show that overexpression of TAZ could protect mitochondrial function.Mitochondrial function damage would cause the cell apoptosis.Therefore,qRT-PCR and Caspase3 activity detection kits were used to detect changes in Caspase3 expression,qRT-PCR was used to detect BCL2 and BAX mRNA changes,and flow cytometry was used to detect changes in apoptosis.The results show that TAZ overexpression could reduce the level of apoptosis in the inflammatory response.In conclusion,overexpression of TAZ could weaken the microglial inflammatory response,reduce the level of cellular oxidative stress,reverse the mitochondrial dysfunction,and ultimately reduce the apoptosis of BV2 cells in the inflammatory response.In order to clarify the anti-inflammatory mechanism of TAZ in microglia inflammation.First,we added NAC pretreatment for 4h,and then added LPS to continue co-treatment.Flow cytometry were used to detect ROS and O2-,and qRT-PCR and ELSIA were used to detect changes in TNF-?,IL-6 and IL-1? expression.It was found that NAC could reduce the level of oxidative stress in the microglia inflammatory response,which in turn weakens the inflammatory response.However,overexpression of TAZ could reduce ROS levels,and it is speculated that TAZ regulates cellular inflammation through ROS.Compared the based sequence of the promoter region of each component of the antioxidant signal pathway,and find the TEAD binding sequence in the Nrf2 promoter.Three reporter gene plasmids were constructed according to the positions of the three TEAD binding sequences which founded in the Nrf2 promoter region.At the same time,the bases of the binding sequence were subjected to base substitution mutation to construct three reporter gene mutant plasmids.The reporter gene activity was detected by the dual luciferase detection kit.The expression of Nrf2 were detected by qRT-PCR and Western blot.The promoter activity of the antioxidant element ARE were detected by the dual luciferase detection kit.The results showed that TAZ overexpression can increase the promoter activity of the first site of Nrf2 and increase the expression of Nrf2 and ARE.After overexpression of TAZ,ML385(the Nrf2 inhibitor)would be added for cotreatment for subsequent experiments.Detection of ROS,O2-,mtROS,mitochondrial membrane potential and apoptosis by flow cytometry;expression of TNF-?,IL-6 and IL-1? by qRT-PCR and ELSIA;detection the changes of I?B,P-I?B and P65 by Western blot;detection changes in Caspase3,GR,SOD,CAT,GPX,GSH / GSSG,MDA and ATP with corresponding detection kits;detection Bax and Bcl2 by qRT-PCR.The results show that after adding ML385,the above protective effect of TAZ is suppressed.After overexpression of TAZ,SOD,CAT,GSH,GPX and GR inhibitors and LPS stimulation were added to detect total cellular ROS;qRT-PCR and ELSIA were used to detect the expression of three pro-inflammatory factors;by Western The expression of p65,p-I?B and I?B was detected by blotting.The results showed that the addition of five antioxidant enzymes and antioxidant inhibitors produced similar effects as the addition of ML385.In summary,TAZ could directly bind to the Nrf2 promoter region through TEAD,increasing the expression of Nrf2 and downstream ARE.The addition of Nrf2 inhibitor ML385 reversed the anti-inflammatory effect of TAZ on microglia during inflammatory response and the protective effects of cellular oxidative stress,mitochondrial dysfunction and apoptosis.Adding the five kind of inhibitors separately could produce the same effect as adding ML385.TAZ could increase Nrf2 expression through TEAD and Nrf2 promoter region,increaseing the SOD,CAT,GPX,GP and GSH expression,reducing the cellular ROS levels,reducing the cellular inflammation,restoring mitochondrial function,and inhibiting the apoptosis.