Designed Fabrication Of Dual Anti-oxidation And Anti-inflammatory Nanozymes For Liver Injury Therapy

Drug-induced acute liver injury（DILI）,especially acetaminophen（APAP,a common antipyretic and analgesic drug）induced liver injury,is the leading cause of acute liver failure in many countries,which is closely associated with reactive oxygen species（ROS）and inflammation.N-acetylcysteine（NAC）is presently the first-choice antidote for DILI,it exhibits therapeutic effects when given at the early stage of ALI.Nevertheless,by the time patients arrive for medical attention,they tend to miss the prime time for NAC treatment.Although liver transplantation could be used as an alternative treatment option in such conditions,the life-long anti-rejection therapy and monitoring of the patient’s condition will definitely be troublesome for patients.Therefore,it is of urgent demand to develop novel therapeutic agents for ALI treatment with long therapeutic time windows.Previous studies have shown that the high levels of reactive oxygen species（ROS）in the injured liver tissues results in oxidative stress and peroxidation,which is one of the significant factor of tissue damage.Therefore,the elimination of ROS contributes to the inhibition of DILI.For example,NAC can significantly reduce the level of oxidative stress by promoting the production of intracellular glutathione（GSH）,achieving the effect of detoxification.In addition,inflammation plays an important role in the disease progression of DILI:in the early stage of liver injury,mononuclear-derived macrophages exhibit pro-inflammatory function,which can aggravate the inflammatory response at the injury site,and thus worsening the liver injury;however,a certain degree of inflammation helps to eliminate necrotic liver cells and promote the repair process of liver injury.Therefore,in addition to the removal of ROS,the regulation of inflammation is also very important in the treatment of DILI.Nanomaterials with catalase（CAT）-mimic and/or superoxide（SOD）-mimic activities can be used to scavenge ROS and generate oxygen,which is beneficial to alleviate inflammation.Particularly,ceria nanoparticels with excellent ROS scavenging capability have been utilized in various biomedical applications,including anti-inflammation therapy,nervous system diseases treatment,wound healing and so on.In this paper,we first designed and synthesized oleylamine-coated ceria nanozymes,and charactered the structure of them by transmission electron microscopy（TEM）,high-reslution transmission electron microscopy（HRTEM）,X-ray diffraction（XRD）pattern and X-ray photoelectron spectroscopy（XPS）.These results showed that ceria nanozymes with diameters of～3 nm had near-spherical morphologies and excellent crystallinities.1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy（polyethylene glycol）-2,000](DSPE-PEG_2K)was used to impart the colloidal stability of the CeNZs in aqueous solution.The TEM and dynamic light scattering（DLS）were employed to analyze the dispersibility and sizes of CeNZs.The results showed that CeNZs had good monodispersity with a size of～12 nm.The surface modification of CeNZs were characterized by infrared spectrometer（FITR）.Furthermore,the catalase-like（CAT）,superoxide dismutase（SOD）and·OH scavenging properties of CeNZs were characterized unsing a specific oxygen electrode on Multi-Parameter Analyzer,total SOD activity detection kit and hydroxyl radical detection kit,respectively.The results show that CeNZs can effectively scavenge ROS including H₂O₂,·O₂^-and·OH.The cytoprotective effects of CeNZs on L02 cells was analyzed by SRB assay.The results showed that CeNZs exhibited protective effects on APAP-treated L02 cells in a dose-depedent manner.Furthermore,the protective effects of CeNZs on liver function were investigated by dectecting the level of serum ALT as well as AST.The results show that the administration of CeNZs into mice 2 h prior to the establishment of the DILI model can effectively reduce the high serum ALT and AST levels induced by APAP.Additionally,after the establishment of the DILI model,CeNZs were simultaneously injected to the mice and the serum ALT and AST levels can also be decreased significantly.Hematoxylin-eosin（HE）staining of liver tissue sections also demonstrated that CeNZs administered to mice 2 h in advance or at the same time can effectively inhibit APAP-induced liver tissue damage.To further evaluate the therapeutic effect of CeNZs on DILI,mice were treated with CeNZs or NAC at 3 h after the establishment of the DILI model,and the liver tissues of mice were havested and evaluated.The results show that NAC has no protective effect on APAP-induced DILI,but CeNZs can significantly reduce the necrosis area of liver tissues and relieve cell death in liver tissues.The protective effect of CeNZs on APAP-induced DILI was investigated by examining the survival rate of mice after different treatments.The results showed that CeNZs can significantly improve the survival rate of mice receiving high-dose of APAP.The molecular mechanism underlying the capability of CeNZs to prevent DILI was further interrogated by flow cytometry,Westren blot,q RT-PCR,and TUNEL staining.The level of ROS in L02 cells was analyzed by flow cytometry;the cytoprotective effects of CeNZs on H₂O₂-treated L02 cells was analyzed by SRB assay.To evaluate the antioxidant effect of CeNZs in vivo,the expression of ROS-related genes in liver tissue was investigated by q RT-PCR.The in vitro results indicate that CeNZs can efficiently eliminate excess cellular ROS and inhibit the toxicity of H₂O₂ to L02 cells.The in vivo results showed that CeNZs participated in the upregulation of Nrf-2 and activated the Nrf-2/Keap1 pathway,and thus regulat the ROS-related genes（including upregulation of Nqo1,Gpx1,HO-1 and downregulation of Nox2,Cyp2e1）,ultimately reducing the ROS levels in liver tissues.In addition,the non-parenchymal cells（NPC）in livers from DILI mice were isolated.The flow cytometry was used to detect the ratio of pro-inflammatory and anti-inflammatory macrophages to investigate the effect of CeNZs on inflammation.At the same time,the regulatory effect of CeNZs on inflammation was further verified on Raw 264.7 cells.The in vivo results showed that CeNZs can significantly reduce the proportion of CD86+proinflammatory macrophages and reduce the expression of inflammatory factors such as Il-1βand TNF-α.At the same time,CeNZs with CAT-like activity can significantly reduce the expression of hypoxia-inducible factor（HIF-1α）in LPS-treated Raw 264.7 cells,and significantly reduce the Il-1βand TNF-αin Raw 264.7cells under hypoxic conditions.In summary,the results above showed that the CeNZs are highly efficient for DILI treatment,which is of great significance for clinical patients who are unconsciously suffering APAP overdose and missing the best time for NAC treatment.Compared with NAC,CeNZs not only scavenge ROS but also alleviate hypoxia via oxygen generation,resulting in the downregulation of the pro-inflammatory cytokines,which facilitates the reduction of the pro-inflammatory macrophages to avoid the secondary damage induced by inflammation.Consequently,for the treatment of DILI,CeNZs show an extended therapeutic time window as compared with NAC,which is ascribed to their ability in the regulation of inflammation.Lastly,this study suggest that CeNZs,with detoxification and anti-inflammation dual regulatory roles,may serve as a promising therapeutic agent in the clinical treatment of ALI,especially for the delayed clinical treatment.