Effect And Mechanism Of Evodiamine Nanoliposomes On Carbon Tetrachloride Or Thioacetamide-induced Liver Fibrosis In Mice

Background:Hepatic fibrosis is a common pathophysiological process of chronic or repetitive liver injury,which is characterized by the damage and death of a large number of hepatocytes,replaced by excessively produced fibrous tissue,accompanied by destruction of normal liver structure and loss of liver function.Persistent liver fibrosis is one of the risk factors for cirrhosis and even liver cancer.Liver transplantation is the only effective method for the treatment of end-stage liver failure of liver fibrosis.However,the limited liver source and high medical cost limit its application.Studies have shown that the occurrence and development of liver fibrosis involves uncontrolled inflammation and oxidative stress,which further stimulates the activation of hepatic stellate cells,resulting in excessive deposition of extracellular matrix.Despite some progress in the study of the pathogenesis of liver fibrosis,there is still a lack of effective therapeutic drugs for it.Therefore,it is particularly important to seek new therapeutic targets and explore effective therapeutic drugs on this basis.Evodiamine is the biologically active alkali component of the plant evodia,which has anti-inflammatory and anti-oxidantive effects.However,evodiamine has extremely poor water solubility,which limits its clinical application.Therefore,in this study,a method for delivering evodiamine using nanoliposomes as a carrier was explored,and its protective and therapeutic effects on liver fibrosis in mice were evaluated.Objective:This study aimed to evaluate the protective effect of evodiamine nanoliposomes on carbon tetrachloride?CCl₄?and thioacetamide?TAA?-induced liver fibrosis in mice and its potential mechanism.Methods:1.Preparation and characterization evaluation of evodiamine nanoliposomesA thin film dispersion method was used to prepare evodiamine nanoliposomes with cholesterol,hydrogenated soybean lecithin?HSPC?and evodiamine as raw materials.The particle size?Size?,potential?Zeta?and polymer dispersity index?PDI?of the evodiamine nanoliposomes obtained by the Malvern particle size analyzer were measured;the appearance was observed with a transmission electron microscope;the infrared spectrum was detected by Fourier infrared detection;the ultraviolet spectrophotometer calculates its encapsulation rate and drug loading;high performance liquid chromatography-mass spectrometry?HPLC-MS?was used to detect the content of evodiamine in the serum and liver of mice to evaluate the bioavailability.2.Protective effect of evodiamine nanoliposomes on mice with liver fibrosisC57BL/6 mice were randomly divided into 6 groups:blank group, evodiamine nanoliposome control group?100 mg/kg??Evo group?,CCl₄model group?CCl₄ group?,TAA model group?TAA group?,CCl₄+evodiamine nanoliposome?100 mg/kg?group?CCl₄+Evo group?,TAA+evodiamine nanoliposome?100 mg/kg?group?TAA+Evo group?,6 in each group.The CCl₄ and CCl₄+Evo groups were intraperitoneally injected with CCl₄,and the TAA and TAA+Evo groups were intraperitoneally injected with TAA,all of which were three times a week for eight weeks.The remaining two groups were intraperitoneally injected with saline as a control.Starting from the fourth week,the CCl₄+Evo group,TAA+Evo group and Evo group were given 100mg/kg intragastric administration of evodiamine nanoliposomes three times a week for five weeks.At the same time,the remaining three groups were given an equal volume of 0.5%sodium carboxymethyl cellulose?CMC-Na?solution.After eight weeks,mouse serum and liver tissue were collected.Liver damage was evaluated by measuring the activities of serum alanine aminotransferase?ALT?and aspartate aminotransferase?AST?and hematoxylin-eosin?HE?staining;Sirius red and Masson staining to observe the degree of fibrosis in mouse liver;To detect the activities of superoxide dismutase?SOD?,glutathione peroxidase?GSH-PX?and the contents of glutathione?GSH?,malondialdehyde?MDA?by kits,and to detect reactive oxygen species by ROS Fluorescent Probe-DHE?DHE?to determine the degree of oxidative stress.To measure the expression of interleukin-6?IL-6?,tumor necrosis factor??TNF-??and CD45 by IHC and immunofluorescence method?IF?to determine the expression of F4/80 to evaluate the degree of inflammation.To detected the expression of activated hepatic stellate cell surface markera-SMA by IHC;Western Blot?WB?was used to detect the protein expression of nucleotide?binding domain,leucine?rich?containing family,pyrin domain?containing?3?NLRP3??apoptosis?associated speck?like protein?ASC??Cleaved-Caspase-1?C-Caspase-1??IL-1b?TGF-??Smad2/3?p-Smad2/3.Results:1.The prepared evodiamine nanoliposomes are spherical and uniform in size.The average particle size of evodiamine nanoliposomes is 97.9±2.61nm,average zeta potential is-35.6±4.23 mV,and polymer dispersity index?PDI?was 0.146,and the drug loading was 29.9%,the encapsulation rate was 84.8%;The content of evodiamine nanoliposome in blood is 834.36%of evodiamine,and the content of evodiamine nanoliposome in liver is789.73%of evodiamine.2.Evodiamine nanoliposome treatment significantly reduced the activities of ALT and AST?P<0.05?and effectively improved the pathological morphology of the liver and reduced the degree of liver fibrosis.Evodiamine nanoliposomes effectively increased the content of GSH and the activities of GSH-PX and SOD?P<0.05?and decreased the content of MDA?P<0.05?and the expression of DHE?P<0.05?.3.Evodiamine nanoliposomes effectively reduced the expression of total leukocyte surface marker CD45 and macrophage surface marker F4/80,and significantly reduced the release of inflammatory factors IL-6 and INF-?,and reduced the expression of activated hepatic stellate cell surface markera-SMA.4.Evodiamine nanoliposomes significantly reduced the protein expression of NLRP3,ASC,C-Caspase-1?IL-1b?TGF-??Smad2/3?p-Smad2/3.Conclusion:Evodiamine nanoliposomes exert its liver protective effects on liver fibrosis through their antioxidant and anti-inflammatory effects,which may be related to regulate the NLRP3/TGF-?/Smad signaling pathway.