Research On The Antioxidant Mechanism Of A Marine Natural Product Based On The Nrf2/ARE Signaling Pathway

Objectives: To investigate the effect of natural compound N-Me-trichodermamide B(compound 1)on the expression of Nrf2,ARE and its downstream phase ?metabolic enzyme HO-1 in Ha Ca T cells by in vitro experiments,and the activity of the upstream pathway was explored.Methods: Firstly,the compounds with low toxicity were screened by CCK-8method.Flow cytometry was used to study the phenotype of oxidative stress cells,and finally a compound with anti-oxidative stress was screened out.Then,the expression of HO-1 in the cells and the effect of Nrf2 protein expression were detected by western blot and RT-q PCR.The nuclear entry of Nrf2 was detected by western blot and immunofluorescence.The ARE plasmid was used for transfection and to measure the ARE luciferase activity.Through the Nrf2 gene silencing experiment,transiently transfected small interfering RNA si-Nrf2 into the cell,after successful transfection,detected the activity expression of Nrf2,ARE,HO-1again,and explored the activity relationship among the three;The expression level of phosphorylated protein kinase p38 was detected;the cells were pretreated with a specific inhibitor of p38 for 4 hours,and then the cells were subjected to the assay,and the expression of Nrf2 and HO-1 was detected.Results: A novel structural monomeric compound N-Me-trichodermamide B(indicated by compound 1)was screened out.The results showed that compound 1significantly induced the expression of HO-1 protein and significantly increased HO-1 The m RNA expression was statistically significant compared with the control group.Subsequently,compound 1 increased the nuclear expression of Nrf2 and up-regulated the activity of the ARE reporter gene.Compared with the control group,the result was statistically significant.The immunofluorescence method also detected that compound 1 could promote the entry of Nrf2 into the nucleus;after the Nrf2 gene was silenced,the expression of HO-1 protein decreased and the activity of ARE decreased.Compound 1 increased the expression of phosphorylated p38 protein.After the p38 specific inhibitor SB239063 was used,the nuclear expression of Nrf2 was decreased,and the expression of HO-1 was down-regulated.Compared with the control group,the results were statisticallysignificant.Conlusion: A natural compound(compound 1)with anti-oxidative stress activity was finally screened and the structure of the compound was not reported.Compound 1 can up-regulate the expression of HO-1 by activating the Nrf2 / ARE signaling pathway,which depends on the activation of phosphorylated p38 protein.