Study On The Molecular Mechanism Of Olaquindox-induced Apoptosis And Cell Cycle Arrest

Olaquindox,a quinoxaline-1,4-dioxide,as an antibacterial feed additive is used to treat bacterial infections in animals and promote animal growth.Olaquindox has been shown to be genotoxic and has been banned in some countries and regions.China also banned olaquindox as a feed additive in 2017.However,due to its low production cost,good antibacterial and growth promoting effects,it is still illegally used in the breeding industry in China.And its molecular mechanism of toxicity is still not clear,olaquindox poses a serious threat to human health.In present study,mouse and HepG2 cells were used to investigate the signal transduction pathways in olaquindox-induced hepatotoxicity,apoptosis,and cycle arrest,and explored the mechanism of curcumin in reducing the toxic effects of olaquindox.The results of this study were divided into the following five sections:(1)We successfully established a mouse model of olaquindox with olaquindox concentrations of 100,200,and 400 mg/kg administered continuously for 28 days.The results showed that olaquindox could significantly induce hepatic oxidative stress and mitochondrial injury in a dose-dependent manner in mice.Olaquindox could induce hepatic injury in mice by activating the mitochondrial apoptotic pathway and activating p53/GADD45a/p21,Nrf2/HO-1,AKT,NF-kB,MAPK signaling pathways and autophagy pathway.(2)In this study,we successfully established a low expression and stable transfection cell line of GADD45a,which proved that GADD45a could regulate olaquindox-induced ROS production and mitochondrial apoptosis pathway.Interference with GADD45a gene increased olaquindox-induced apoptosis and DNA damage,further activating the JNK/p38 pathway.Inhibition of JNK/p38 pathway by JNK inhibitor(SP600125)and p38 inhibitor(SB203580)significantly inhibited GADD45a activation and increased olaquindox-induced apoptosis,DNA damage,and S-phase arrest,suggesting that the JNK/p38 pathway played protective roles in olaquindox-induced apoptosis.At the same time,the JNK/p38 pathway was partially involved in GADD45a-regulated olaquindox-induced apoptosis and S-phase arrest.(3)In this study,we successfully established a low expression and stable transfection cell line of p21.Olaquindox inhibited the expression of p21 protein in a p53-independent manner.AKT phosphorylated p21 and induced p21 protein transfer from the nucleus to the cytoplasm,which played an anti-apoptotic role.AKT and Nrf2/HO-1 signaling pathways were involved in olaquindox-induced HepG2 mitochondrial apoptosis and S phase arrest,further validating the experimental results in mice.PI3K/AKT inhibitor(LY294002)and Nrf2/HO-1 inhibitor(ZnPP-?)inhibited AKT pathway and HO-1 signaling pathway,increased olaquindox-induced apoptosis and cycle arrest,indicating that the inhibition of AKT and Nrf2/HO-l signaling pathway played pro-apoptotic roles.Interference with p21 gene increased olaquindox-induced mitochondrial apoptosis and S phase arrest through further activating the AKT pathway,inhibiting of Nrf2/HO-1 signaling pathway.(4)The regulation of GADD45a and p21 in olaquindox-induced cytotoxicity was mainly p53-independent.Further studies confirmed that p53 played a pro-apoptotic role in olaquindox-induced toxicity,p53 knockdown reduced olaquindox-induced mitochondrial apoptosis,and p53 gene deletion mouse model further validated the pro-apoptotic effects of p53.p53 may exert its pro-apoptotic role by inhibiting the autophagy pathway and activating the JNK/p38 signaling pathway.(5)Mouse and cell models demonstrated that curcumin significantly reduced olaquindox-induced oxidative stress and intracellular ROS levels,caspase acti,vation,and apoptosis.Curcumin enhanced its antioxidant capacity by up-regulating the Nrf2/HO-1 pathway,down-regulating the NF-kB pathway and inhibiting NF-kB nuclear translocation.Further studies confirmed that curcumin could improve olaquindox-induced liver injury and mitochondrial apoptosis through activating the Nrf2/HO-1 pathway,inhibiting NF-kB and p53 pathways.In summary,the present study evaluated the toxicity of olaquindox in vivo and in vitro,which confirmed that GADD45a and p21 regulated olaquindox-induced apoptosis and cell cycle arrest in a p53 independent manner,while JNK/p38,AKT,and Nrf2/HO-1 pathways involved in regulation of olaquindox-induced apoptosis and cell cycle arrest.This study provides a scientific basis for assessing the safety of olaquindox and other quinoxalines for food animals and humans,and provides a theoretical basis for the clinical use of veterinary drugs and the development of new veterinary drugs.