Chrysin Liposomes Ameliorated Hepatic Ischemia-reperfusion Injury In Mice

Objective This study amied to explore the protective effect of chrysin liposomes on hepatic ischemia-reperfusion injury in mice and its potential mechanism.Methods1.Preparation and evaluation of chrysin liposomesA thin film dispersion method was used to prepare chrysin liposomes with cholesterol,lecithin and chrysin as raw materials.The particle size,zeta potential and polymer dispersity index(PDI)of the chrysin liposomes obtained by the Malvern particle size analyzer.The morphology of chrysin liposomes were measured by transmission electric microscope.The drug loading and encapsulation efficiency of chrysin liposomes were determined by ultraviolet spectrophotometer.The content of chrysin in the serum and liver of mice were detected by ultra performance liquid chromatography-mass spectrometry.2.To explore the effect of chrysin liposomes on hepatic ischemia-reperfusion injury in mice and its potential mechanism.The male C57BL/6J mice were randomly divided into 4 groups,shamoperation group(Control group),liver ischemia-reperfusion model group(HIR group),liver ischemia-reperfusion model + chrysin liposomes group(HIR + CL group),chrysin liposome group(CL group),six mice in each group.The mice of HIR + CL group and CL group were intragastric administrated with chrysin liposomes(100 mg/kg)at 16.5 h,8.5 h,0.5 h before surgery,while Control group and HIR group were intragastric administrated with same volume 0.9% saline.The non-invasive arterial clamp was used to block the flow of left and middle lobe of the liver,resulting in ischemia of the entire 70% of the liver tissue,and the arterial clamp was removed 90 min later to restore blood reperfusion.The blood was collected after reperfusion for 6 h,and liver tissue was harvested after reperfusion for 24 h.The level of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum were determined by the commercial kit.Hematoxylin and eosin(HE)stainning was used to observe the morphological changes of liver tissue.The level of oxidative stress in liver tissue were determined by dihydroethidium(DHE)stainning and the content of malonaldehyde(MDA).Deoxyribonucleotide terminal transferase-mediated terminal labeling method(TUNEL)was used to detect hepatocyte apoptosis.Immunohistochemistry(IHC)was used to test the expression of the TNF-α and IL-6.Immunofluorescence(IF)staining was used to detect the infiltration of inflammatory cells in liverparenchyma.The western blot(WB)was used to detect the protein expression of Bcl-2,BAX,NLRP3,ASC,C-Casp-1,C-Casp-3,IL-1β.Results1.The morphology of the chrysin liposomes was spherical or nearly spherical.And the average particle sizes of chrysin liposomes was 129±13.53 nm,the PDI was 0.265±0.021,the zeta potential was-34.46 ± 4.14 mV,the encapsulation rate was 95.03±2.17%,and a drug loading was 16.4±0.8%.The content of chrysin liposomes in serum is 2.54-fold of chrysin,and the content of chrysin liposomes in liver is 1.45-fold of chrysin.Compared with the Control group,the levels of ALT and AST in serum were significantly increased in the HIR group.The hepatic sinusoids were congested significantly,and a large amount of focal necrosis were observed in the HIR group.The number of apoptotic hepatocytes in the HIR group were notably increased compared with the Control group.The infiltration of inflammatory cells and the expression of IL-6 and TNF-α in liver tissue of HIR group were increased obviously as compared to the Control group.Compared with the Control group,the number of DHE-positive cells and the content of MDA in the HIR group were increased significantly.In the HIR group,the expression of C-Casp-3,BAX,NLRP3,ASC,C-Casp-1,IL-1β in liver tissue were increased and the expression of Bcl-2 was decreased,which compared to the Control group.After chrysin liposomes pretreatment,compared with the HIR group,the serum ALT and AST activity levels of mice were significantly reduced,and the hepatic sinusoids congestion as well as the area of focal necrosis were reduced,and the number of apoptotic hepatocytes were notably decreased.The infiltration of inflammatory cells and the expression of IL-6and TNF-α in liver tissue of HIR+CL group were decreased obviously as compared to the HIR group.Compared with the HIR group,the number of DHE-positive cells and the content of MDA in the HIR+CL group were declined significantly.In addition,the expression of C-Casp-3,BAX,NLRP3,ASC,C-Casp-1,IL-1β in liver tissue of HIR+CL group were decreased and the expression of Bcl-2 was increased compared with the HIR group.Conclusion1.The liposomes can be successfully prepared by the thin film dispersion method,using lecithin and cholesterol as membrane materials.The chrysin liposomes can significantly increase the relative bioavailability of the chrysin and increase the drug concentration in the liver.2.Chrysin liposomes alleviate liver ischemia-reperfusion injury through its anti-inflammatory,anti-oxidation,anti-apoptotic effects.3.The protective effect may be related to suppress the activation of NLRP3 inflammasome and downregulate the expression of NLRP3,ASC,C-Casp-1 and IL-1β.