Study Of The Protective Effect Of 8-Octyl Berberine On Acute Liver Failure And Its Mechanism

Acute liver failure（ALF）is a clinical syndrome accompanying severe hepatocyte necrosis and hepatic failure and with high mortality clinically.But so far there is no available treatment for ALF except for liver transplantation.Therefore,the search for a safe and effective prevention method is particularly essential.The aim of present study was to investigate the hepatic protection effect and potential molecular mechanism of8-octyl berberine in Kunming mice and L02 cells,providing a theoretical basis for the prevention of acute liver injury.The main contents and results of this study are as follows:（1）We investigated the safety of 8-octyl berberine.The acute toxicity test of kunming mice showed that the LD₅₀0 value of 8-octyl berberine was about 720.138mg/kg.In the sub-chronic toxicity study of SD rats,there was no significant changes in the important organ index（heart,liver,spleen,lung,kidney）and hematological analysis in 8-octyl berberine-treated rats compared to the control group.Therefore,8-octyl berberine is a relatively safe compound.（2）In vivo experiments,the protective effect of 8-octyl berberine on acute liver failure was studied using D-galactosamine（D-GalN）/lipopolysaccharide（LPS）-induced acute liver injury mice model.We observed the effect of 8-octyl berberine on the survival rate of mice induced by D-GalN/LPS,observed the pathological changes in liver tissue,and examined the activities of alanine transaminase（ALT）and aspartate transaminase（AST）.The levels of tumour necrosis factor-α（TNF-α）,interleukin-6（IL-6）and C-reactive protein（CRP）in serum,and the expressoin of cyclooxygenase-2（COX-2）,inducible nitric oxide synthase（iNOS）in liver tissue were detected.Furthermore,the glutathione（GSH）and malondialdehyde（MDA）levels and superoxide dismutase（SOD）activity of liver tissue were also detected.The experimental results showed that 8-octyl berberine markedly improved the survival rate of mice induced by D-GalN/LPS,significantly improved the pathological changes of liver tissue caused by D-GalN/LPS,and markedly reduced the activities of ALT and AST in serum of mice induced by D-GalN/LPS.8-octyl berberine also markedly attenuated the MDA and GSH levels and SOD activities in liver tissue of acute liver injury mice induced by D-GalN/LPS.In addition,8-octyl berberine significantly decreased the levels of TNF-α,IL-6 and CRP in serum of D-GalN/LPS-induced acute liver failure mice and markedly inhibited the production of iNOS and COX-2 in liver tissue of mice induced by D-GalN/LPS.These results consistently showed that 8-octyl berberine has a protective effect on acute liver injury.（3）The potential targets of 8-octyl berberine were predicted based on the principles of network pharmacology and reverse pharmacophore matching methods.The“drug-target-disease”network was constructed through the computer system biological method and network visualization software.The results indicated that the pharmacological action of 8-octyl berberine was concentrated on metabolic diseases,cancer,immune diseases,at the same time,8-octyl berberine was significantly related with various hepatic diseases and inflammation.The results of KEGG pathway analysis and the interaction between receptor and ligand showed that 8-octyl berberine may protect acute liver failure by inhibiting MAPK and NF-κB signaling pathways.（4）In vitro experiments,we explored the mechanism of hepatic protection of8-octyl berberine using LPS-induced L02 cells as an acute liver failure model.Firstly,the effect of 8-octyl berberine on the proliferation of L02 cells was analyzed by MTT assay.The results showed that treatment with 8-octyl berberine within 0.1μg/μL for 24h revealed no obvious influence on the viability of L02 cells,and more than 85%of cells were survived.8-octyl berberine exhibited cell cytotoxicity at high concentrations(IC₅₀0 was 1.28μg/mL).We found that 8-octyl berberine decreased the expression of cytokines such as TNF-αand COX-2 in LPS induced L02 cells in vitro experiments,this indicated that 8-octyl berberine has protective effect on LPS induced L02 cells.The expression of the signaling pathway-related genes in the LPS-induced L02 cells was analyzed by qRT-PCR.The results showed that 8-octyl berberine inhibited the mRNA expression of TLR4,ERK,p38,JNK and NF-κB in LPS-induced L02 cells.In addition,western blot method was used to analyze the influence of 8-octyl berberine on related proteins in the signaling pathway.The results showed that 8-octyl berberine inhibited the protein expression of TLR4,p-ERK,p-p38 and p-JNK in LPS-induced L02 cells,and suppressed the translocation of NF-κB from cytosol into the nucleus in LPS-induced L02 cells.In summary,8-octyl berberine has a good protective effect on acute liver 8-octyl berberine,which could be a potential prevention agent in attenuating acute liver failure.