Experimental Study Of Peptide Drug Development Based On Intracellular Targeted Degradation Of Keap1 To Promote Antioxidant,Anti-inflammatory,and Inhibit Hepatic Stellate Cell Activation

Objective The Nrf2 signaling pathway is a core factor regulating redox balance and an important protection system against exogenous stimuli in the organism.Studies have shown that a large number of oxidative stress and induced signal transduction are involved in the occurrence and development of liver fibrosis.During the activation of hepatic stellate cells,the induction of Keap1 degradation promotes the activation of the Nrf2 antioxidant signaling pathway,thereby providing new ideas and experimental basis for liver fibrosis treatment.Methods(1)Design and synthesize fusion peptide(KKP)based on PROTAC technology and test its transmembrane function in HSC-T6 cells;(2)Western bloting detection to detect whether KKP can degrade the total protein content of Keap1 in HSC-T6 cells(3)Analysis of KKP anti-oxidation response element ARE and NF-κB corresponding response element reporter gene expression by luciferase reporter gene technology;(4)Western bloting detection of KKP Nrf2 downstream antioxidant genes in HSC-T6 cells Effect of protein expression of NQO-1 and NF-κB pathway related genes IL-6;(5)Western bloting was used to detect the effect of KKP on type I collagen expression in HSC-T6 cells.Results(1)Three fusion peptides(KKP)with high affinity(KKP1),low affinity(KKP2),and non-affinity(KKPs)were designed and synthesized based on PROTAC technology,and three fusion peptides KKP penetrated the membrane in HSC-T6 cells.It works well;(2)KKP1 can recruit Keap1 protein in HSC-T6 cells to induce its ubiquitination degradation;(3)KKP1 can promote the expression of genes regulated by ARE response elements in HSC-T6 cells,and promote GCLC,a Nrf2 downstream antioxidant molecule And NQO-1 protein expression;(4)KKP1 can inhibit gene expression regulated by NF-κB response elements in HSC-T6 cells,and down-regulate IL-6 protein expression;(5)KKP1 can up-regulate Col1α2 in HSC-T6 cells Protein expression.Conclusion The fusion peptide KKP1,which contains a short peptide with high affinity to the Keap1 binding domain,can penetrate the membrane into the cell,recruit Keap1 protein to induce its ubiquitination and degradation,thereby release Nrf2 into the nucleus,promote the transcriptional activity of the Nrf2/ARE pathway,and activate the downstream resistance of Nrf2 Oxidative factors,while inhibiting the NF-κB inflammatory signaling pathway and its downstream inflammatory factors,play a role in inhibiting the activation of hepatic stellate cells.It is suggested that the fusion peptide KKP1 based on PROTAC technology may provide new ideas for the treatment of liver fibrosis.