Screening Of Flavonoids Targeting Nrf2 And Its Protective Effect And Mechanism Against Drug-induced Liver Injury

Aceta minophen(APAP)is one of the most widely used antipyretics and analgesics,causing liver damage(DILI)after an overdose.APAP can produce ROS during liver injury,causing oxidative stress,which is the main cause of DILI,inhibiting oxidative stress can reduce APAP-induced liver damage,while flavonoids have good antioxidant capacity and low toxicity characteristics,beco ming the preferred natural product for the treatment of DILI.Therefore,we screened the small molecule library of flavonoids through designed in vitro models and reporter gene experiments,and finally screened the flavonoid compound with strong antioxidant energy,Okanin(Oka).Oka is one of the most abundant flavonoids found in the order Campanula(Asteraceae)and has significant anti-inflammatory and antioxidant activities.This article aims to explore the underlying mechanism of Oka-mediated acute liver injury induced by APAP.Through the results of the CCK-8 experiment,we observed that Oka has a protective effect against APAP-induced liver damage.Western blot results showed that pretreatment with Oka further increased the expression of antioxidant enzymes prior to APAP ad ministration.Furthermore,we observed that the protective effect was associated with upregulation of Nuclear factor erythroid2-related factor 2(Nrf2).Molecular docking results suggest that Oka may be an effective Nrf2 activator to alleviate APAP-induced liver injury by mediating Nrf2 pathway activation.According to the results of immunoblotting and immunofluorescence experiments,Oka activates Nrf2 into the nucleus,binds to the intranuclear antioxidant element AER(Antioxidant response element),and transcribes downstream antioxidant proteins to reduce APAP-induced liver damage.To verify this result,we obtained a stable Hep G2 cell line that knocks down the Nrf2 gene by infecting Hep G2 cells with lentivirus,Hep G2-sh-Nrf2 blocking the protective effect of Oka against APAP-induced liver injury,as well as Nrf2 nucleation.These data provide new insights into the mechanism by which Oka regulates APAP-induced liver injury by targeting Nrf2.