Target Discovery Of Natural Product Britanin And Its Neuroprotective Effect

1.Research purposesThe rising level of reactive oxygen species will break the redox balance state,causing oxidative stress response.Oxidative stress plays an important role in the development and progression of a series of diseases such as neurodegenerative diseases,tissue organ damage and autoimmune diseases.In the course of cerebral ischemia-reperfusion,the level of reactive oxygen species(ROS)in brain tissue increased significantly,which could cause brain injury.Therefore,it is an important way to treat stroke by improving the body's antioxidant level to reduce oxidative stress.Nrf2 signaling pathway is one of the most important ways to resist oxidative stress.By activating the Nrf2 pathway,it enhances the ability of the body to resist oxidative damage.Thus,activation of the Nrf2 signaling pathway becomes a treatment for multiple degenerative diseases and chronic diseases.Targeting the Nrf2 signaling pathway is a new direction for the development of drugs for the treatment of neurodegenerative diseases and chronic diseases.Natural products are rich in sources,with a large pharmacological development potential.Looking back on the history of drug research,natural products have played a pivotal role,contributing a number of indispensable drugs.The natural product Britanin is a guaiacane-type sesquiterpene lactone,widely distributed in the genus Inula plants.In this study,the neuroprotective effect of Britanin was studied by constructing an in vitro model of neuronal injury.In order to elucidate the mechanism of neuroprotective effect,we conducted a study on the regulation of oxidative stress and Nrf2 signaling pathway by Britanin.It is expected to find the molecular target of Britanin in Nrf2 pathway regulation.Ultimately,the ability of Britanin to play a neuroprotective effect in vivo was evaluated by constructing an experimental rat model of cerebral ischemia-reperfusion injury(MCAO).Based on the above studies,we have laid the preclinical research foundation for the development of Britanin into a novel therapeutic drug for oxidative stress-related diseases.2.Research methodology(1)Construction of neural cell injury model in vitro.Two models of in vitro nerve cell injury were constructed.Respectively,glutamate-induced PC12 cell injury model;oxygen and glucose deprivation reperfusion(OGD-R)induced primary cortical neuronal injury model.On the basis of these two cell models,the neuroprotective effect of the natural product Britanin was studied.(2)To study the effect of Britanin on Nrf2 signaling pathway.Natural products Britanin can protect nerve cells and reduce oxidative stress-induced cell damage.In this study,we investigated the effect of Britanin on the antioxidant signaling pathway of Nrf2 at the level of primary neuron cells by ARE reporter gene test,siRNA interference assay,real-time quantitative PCR assay and protein immunoblotting assay.(3)Verify the covalent binding of Britanin to the target protein Keap1.The Keap1 protein is a negative regulatory protein of the Nrf2 protein,which causes its ubiquitination and programmed degradation.In this study,Escherichia coli prokaryotic expression system was used to express and purify human Keap1 full-length protein and its BTB domain.In this study,the biotinylated modified Britanin small molecule probe was designed and synthesized.In the study of in vitro mechanism,we used the small molecule probe pulldown target protein Keap1,initially confirmed the interaction between Britanin and Keap1.In order to further confirm the binding site between Britanin and the target protein Keap1,the effect of Britanin on the cysteine modification in the Keap1 protein sequence was studied by LC-MS-MS,and the covalent binding site was determined.In order to clarify the binding mode of Britanin and the BTB domain of Keap1 protein,we carried out the co-crystallization experiment of Britanin complex with the target protein Keap1.In order to elucidate the molecular mechanism of Britanin in activation of Nrf2 signaling,we studied the regulation and regulation mechanism of Britanin on ubiquitination of Nrf2.(4)The rat MCAO model was constructed in vivo.In order to study the effect of Britanin on the anti-oxidative and neuroprotective effects in the animals,we constructed the MCAO model in rats in vivo,which was correspond to OGD-R-induced primary cortical neuron injury model in vitro.3.ResultsIn the in vitro neuronal protection experiments,we found that Britanin can significantly protect nerve cells,reduce oxidative stress-induced cell damage.At the same time,we found that Britanin can significantly down-regulate the level of ROS in the stimulation of nerve cells.Nrf2 signaling pathway is the most important regulatory pathway for the body to resist oxidative stress and maintain the redox steady state.Through the reporter gene experiment and gene interference experiment,we found that Britanin can significantly activate the Nrf2 signaling pathway,improve the expression of antioxidant enzymes,and then play a neuroprotective effect.The Keap1 protein is a negative regulator of the Nrf2 signaling pathway and may be the target of Britanin.Using the the small molecule probe to conduct pull-down experiment,we confirmed the interaction between Britanin and the target protein Keap1.The covalent binding site between Britanin and Keap1 were further investigated by LC-MS-MS.The cocrystallization structure of Britanin and Keap1 protein BTB domain was obtained by cocrystallization experiment.It was confirmed that Britanin was covalently bound to the 151-position cysteine binding mode of Keap1 by Michael addition.By further molecular mechanism studies,we found that Britanin,by targeting the Keap1 protein,interferes with the interaction between Keap1 and Cul3,which interfere the ubiquitination and proteasome degradation of the Nrf2 protein,thereby activating the Nrf2 signal pathway.Using the model of acute cerebral ischemia-reperfusion injury in rats,we investigated the ability of Britanin to enhance the body's anti-oxidative stress and neuroprotective effect in vivo.Britanin administration can activate the Nrf2 pathway,significantly reduce the infarct volume and improve the behavioral score.4.ConclusionsThis subject found that the natural product Britanin activates the Nrf2 signaling pathway by targeting the Keap1 protein.Britanin is a new Nrf2 signaling pathway agonist.The binding mode of Britanin and Keap1 protein was revealed by the analysis of co-crystal structure,and the molecular mechanism of Britanin 's activation of Nrf2 signaling pathway was elucidated.We found that Britanin could significantly improve the level of antioxidant by activating the Nrf2 signaling pathway,and then play a neuroprotective role in the model of neuronal cell protection in vitro and model of cerebral ischemia-reperfusion injury in rats in vivo.In the follow-up study,by structural modification of Britanin and screening analogues,it is hopeful to find new leading compounds with better activity and better selectivity,and to develop into a new treatment for oxidative stress-related diseases.