Targeting The CO-HIF-1 Axis By Nanoformulation Of CO Donors As A Therapeutic Strategy For Metabolic-associated Fatty Liver Disease

Background Metabolic-associated fatty liver disease(MAFLD)is one of the major classes of chronic liver diseases including steatohepatitis,cirrhosis,and liver cancer.However,currently no drugs are approved for MAFLD.Hypoxia-inducible factor-1α(HIF-1α)has been recognized as an early critical element in the development of MAFLD.Our previous studies indicated that the nanodesigned carbon monoxide(CO)donor styrene maleic acid copolymer(SMA)micelleencapsulating CO-releasing molecule(SMA/CORM2)achieved a potent protective effect in many inflammatory diseases in which suppression of HIF-1αplays an essential role.Objective:In this study,we used a MAFLD model induced by a high-fat methionine-and choline-deficient(HF-MCD)diet to verify CO-HIF-1αaxis may become a modulator and therapeutic target of MAFLD,and to investigate the validity and applicability of CO treatment with SMA/CORM2 for MAFLD,and to explore the mechanisms of action of this treatment.Methods:This study was completed in vivo and in vitro.(1)Adult healthy male ICR mice(8 weeks,34–36 g)were chosed.Under a 12h light and dark cycle,animals are maintained at 20–25°C and 50±5%humidity.The mice were free to eat feed and water and observe the growth state of mice.Mice were fed adaptively for 1 week before experiments.During experiments,the mice were randomly divided into different groups as indicated.To establish the MAFLD model,the mice were fed an HF-MCD diet for 4weeks.In the healthy control group,a standard diet was used instead of the HF-MCD diet.For the SMA/CORM2 treatment groups,SMA/CORM2 of different concentrations(1,3,and 10 mg/kg)was administered intravenously(i.v.)three times per week,and saline was injected i.v.into some mice as untreated controls.On day 29 after the first feeding of the HF-MCD diet,mice were killed after being fasted overnight and body weights were measured.Collectting blood samples from the inferior vena cava,and obtaining the serum subsequently after centrifugation was used for the biochemistry assay as described below.The liver,subcutaneous white adipose tissue(s WAT),gonadal white adipose tissue(g WAT),and perirenal white adipose tissue(p WAT)were collected and weighed.Parts of the tissues were used for Western blotting and real-time reverse transcription-polymerase chain reaction(RT-PCR),and parts of liver tissues were fixed for histological examination and Oil Red O staining.Objective To study a significantly protective and therapeutic effect was achieved by SMA/CORM2 treatment.In some experiments,hydrolyzed SMA,inactivated SMA/CORM2(i SMA/CORM2)that was decomposed by incubation in dimethyl sulfoxide(DMSO)at room temperature for 24 h to completely liberate CO,and native CORM2 that was dissolved in DMSO for i.p.injection were used as controls.The purpose is to eliminate the influence of non-SMA/CORM2 carrier components on MAFLD and determine the role of CO molecule.At the same time,compare the bioavailability of SMA/CORM2 and free CORM2.(2)Mouse hepatocyte AML12 cells were cultured in DMEM:F12 medium supplemented something.To induce lipid accumulation in hepatocytes,AML12 cells were seeded in 6-well plates(1.65×10~5cells/ml),and then after overnight preincubation,high concentrations fatty acids(HF,sodium oleate/sodium palmitate,200μM)were added.At 24 and 48 h after HF treatment,CCK8 reagent was added to evaluate cell viability.By using the above-described in vitro hepatic steatosis model,the effect of SMA/COMR2 was investigated,with indicated doses of SMA/CORM2 were added to cells after HF administration.After scheduled times,collectting cells,proteins and total RNA were extracted by Western blotting and real-time RT-PCR,respectively,as described above.In some experiments,HIF-1αsh RNA and HO-1-specific si RNA were used to knock down HIF-1αand HO-1,respectively,accordingly to the manufacturer’s instructions.Result:(1)These findings of study found marked lipid accumulation in the liver after feeding the HF-MCD diet for 4 weeks,accompanied by dysfunction of lipid metabolism,macrophage polarization toward the pro-inflammatory M1 phenotype,activation of the NLRP3 inflammasome,and activation of the TFGβfibrosis signaling pathway.Up-regulation of HIF-1αis the early and upstream event of these pathological changes,and administration of SMA/CORM2(10 mg/kg,three times per week)significantly suppressed the expression of HIF-1αfrom soon after the HF-MCD diet was eaten when the pathological changes were not apparently seen.Consequently,a significantly protective and therapeutic effect was achieved by SMA/CORM2 treatment.(2)An invitro study with hepatocytes treated with a high concentration of fatty acids showed similar results,and knockdown of HIF-1αby using short hairpin RNA(sh RNA)produced similar effects as SMA/CORM2.(3)In addition,we found that intrinsic CO/HIF-1αmay serve as a feedback loop under physiological conditions.Conclusion:In this study,we determined the therapeutic potential of the nano-designed CO donor SMA/CORM2 in HF-MCD diet-triggered hepatic steatosis and liver fibrosis.The effector molecule in this process was the gasotransmitter CO released from SMA/CORM2,which caused significant suppression of HIF-1α,a central occurrence in the development of MAFLD;subsequent inhibition of macrophage M1 polarization and activation of the NLRP3 inflammasome;and restoration of lipid metabolic dysfunction.These findings strongly suggested a new therapeutic strategy for the treatment and/or prevention of MAFLD by using exogenous CO,i.e.,SMA/CORM2,to target the CO-HIF-1α axis.