The Role Of CO-mediated Polarization Of M2 Macrophages In Liver Cell Regeneration In APAP-induced Acute Liver Injury

Background:Acetaminophen(APAP)induced liver injury is the most common drug-induced liver injury,accounting for the top cause of acute liver failure in the United State,however the therapeutic options for it is very limited.Excess generation of reactive oxygen species(ROS)and the subsequent inflammatory responses are the major factors of the liver injury after APAP.Carbon monoxide(CO)is an important gaseous molecule with versatile functions including anti-oxidation and anti-inflammation,and we previous reported the therapeutic potential of a nano-designed CO donor SMA/CORM2 in a dextran sulphate sodium(DSS)induced mouse colitis model.In this context,we investigated the effect of SMA/CORM2 in an APAP-induced mouse acute liver injury model and tackled the mechanisms involved.Objective:In order to explore the protective effect and mechanism of nano-designed CO donor SMA/CORM2 in APAP-induced acute liver injury.Methods:This experiment was divided into animal experiments and in vitro experiments.Animal experiment:Adult healthy male ICR mice(34-36 g)were purchased from Beijing Weitong Lihua Company(Beijing,China),and some mice for in vivo CO measurement were from SLC,Shizuoka,Japan.The animals were maintained at 20-25℃and 50±5%humidity with 12 h light and dark cycle.All experiments were approved by the Medical Ethics Committee of Anhui Medical University and Animal Ethics Committees of Sojo University,and carried out according to the Laboratory Protocol for Animal Handling of Anhui Medical University and Sojo University.Mice were fed adaptively for 1 week before experiments,allowing the animals to drink and eat freely.During experiments,the mice were randomly divided into different groups as indicated,and the mice were fasted for 12 hours before administration of drugs or agents.APAP-induced liver injury model was established by intraperitoneal(i.p.)injection of a single dose of 300 mg/kg APAP,in the SMA/CORM2treatment group,SMA/CORM2 was injected intravenously(i.v.)through the tail vein 1h after APAP administration.The mice were sacrificed and weighed within a predetermined time,and the dose-effect(1,5,25 mg/kg)and time-effect relationship(0,2,4,24,48h)were established.Vitro experiments:Macrophages RAW264.7 were seeded in 6-cm petri dish,after overnight preincubation,different concentrations of SMA/CORM2(0,0.01,0.025,0.05,0.1mg/ml)were added.After scheduled time(0,2,4,24,48h),medium was removed and cells were collected,and observe the effect of CO on macrophage polarization;Macrophages RAW264.7 were seeded in 6-cm petri dish,after overnight preincubation,CO donor SMA/CORM2(0.1 mg/ml)were added.After scheduled time(24h),medium was collected as conditioned medium.AML12cells were seeded in 6-cm petri dish(1.5*10~5 cells/well)or 96-well plate(5000cells/well),after overnight preincubation,medium was removed and above-mentioned CM were added,after further 24 h and 48 h incubation,observe the effect of CO-induced macrophage polarization on hepatocyte proliferation.Result:(1)This project observed the dynamic changes of HIF-1α,HO-1 and macrophage polarization in the APAP-induced acute liver injury model,and found that the expression of HIF-1αand HO-1 and the polarization of macrophages were different at different time points of APAP-induced liver injury.HIF-1αis highly expressed in the early stage of liver injury;The expression of HO-1 increases when liver injury was severe,and played a protective role;Macrophages were mainly polarized to M1 type in the early stage of liver injury,and mainly to M2 type in the late stage of liver injury.And it was confirmed that CO can also induce macrophages to polarize to M2 type in vitro experiments.(2)Injection of CO donor SMA/CORM2 through the tail vein after 1h after APAP,serum ALT,AST,and liver GSH,MDA were significantly reduce,and effectively protect APAP-induced acute liver injury.(3)After the administration of SMA/CORM2,the blood and liver CO content of mice increased significantly.(4)Injection of CO donor SMA/CORM2 through the tail vein after 1h after APAP,and found that the polarization of M1 macrophages was significantly inhibited,and the expression of M2 macrophages was significantly increased.(5)It was found that HIF-1αwas significantly inhibited after injection of CO donor SMA/CORM2 through the tail vein after 1h after APAP.(6)Injection of CO donor SMA/CORM2 through the tail vein after 1h after APAP,and found that CO can activate the Pi3k/Akt/m TOR signaling pathway and promote the proliferation and regeneration of liver cells.Conclusion:This study found that CO released from SMA/CORM2 regulated the reprogramming of macrophages into M2 type by inhibiting HIF-1α,thereby protecting APAP-induced acute liver injury.Therefore,we expected that SMA/CORM2 can be used as a treatment option for APAP-induced liver injury and other inflammatory diseases.