Hepatoprotective Effects Of Triterpenoids Extracted From Antrodia Cinnamomea Fermentation Mycelia In Mice Models With Alcohol-induced Liver Injury

Antrodia cinnamomea,a precious,rare and unique fungus that has been used as a folk medicine in Taiwan for centuries is known to relieve drunks and hepatoprotective effects.Triterpenoids are important active compounds of A.cinnamomea.With a progressively alcohol consumption,alcoholic liver disease has become an public health problem all over the word and is urgently needed to be resolved.Hepatoprotective effects of triterpenoids extracted from Antrodia cinnamomea mycelia(ACT)and the its underlying mechanisms were explored using a mouse model of acute and chronic alcohol-induced liver injury.In the present study,the components of A.cinnamomea mycelia(AC)were systematically analyzed.AC contained 8.05% triterpenoids,25 types of fatty acid,16 types of amino acid,3 types of nucleotide,and 8 types of mineral.Lead(Pb),mercury(Hg),arsenic(As),and cadmium(Cd)were not detected,and the concentration of cuprum(Cu)is much lower than the official safety limits for humans.The hepatoprotective effects were observed after 2 weeks of AC treatment in the mouse model which is established by 9.52 g/kg white spirit(an wine with 56°).Results demonstrated that AC administration significantly reduced the ratio of aspartate aminotransferase/alanine aminotransferase(AST/ALT),and against alcohol-induced damage have been directly certified by reducing necrosis,inflammatory infiltration,fatty droplets formation and apoptosis rate in liver.This may be associated with protein kinase B/nuclear factor kappaB(Akt/NF-?B)signaling.The univariate methods were used to optimize the ethanol extract of crude triterpenoids from A.cinnamomea mycelia.The optimum extration process was described as: A.cinnamomea mycelia was extrated with 70% ethanol,under 80? with the liquid-solid ratio of 20:1 for 100 min.The candidate component ACT was purified from A.cinnamomea mycelia ethanol extract with AB-8 type of macroporous resin column.The liquid chromatograph mass spectrometer(LC-MS)and LC-MS/MS were performed to determine the types of ACT.ACT mainly contains 25 types of triterpenoid compounds.C57BL/6 mice with acute alcohol-induced liver injury were induced by oral gavage of 13 g/kg white spirit(an wine with 56°).Two-week alcohol consumption caused significant weight loss,liver dyslipidemia and the elevation of alanine aminotransferase,aspartate aminotransferase,?-glutamyl transferase and alkaline phosphatase activities in serum and/or liver,which were markedly reversed after two-week ACT administration.ACT alleviated the organ structures changes and inflammatory infiltration of alcohol-damaged tissues,and reduced hepatocyte apoptosis.ACT inhibits the pro-inflammatory cytokines levels(nitric oxide,reactive oxygen species,tumor necrosis factor alpha,interferon,interleukin 1?,7 and 33,regulate upon activation normal T cell expressed secreted,P-selectin,chemokine(C-X-C motif)ligand 13,human cartilage glycoprotein 39,plasminogen activator inhibitor-1,vascular endothelial growth factor)and enhancing anti-inflammatory cytokines(interleukin 22).Acute excessive alcohol promoted inflammation with significant correlations observed with hypoxia-inducible factor 1 alpha(HIF-1?),which was strongly reduced by ACT via its modulation on the Akt/70-kDa ribosomal protein S6 kinase phosphorylation(p70S6K)and Wnt/?-catenin signaling.The hepatoprotective effects of ACT in mouse with chronic alcohol-induced liver injury was established with white spirit for 24-week.The hepatoprotective effects of ACT have been confirmed by decreases in ALT and AST levels in liver.ACT alleviated dyslipidemia by decreasing the levels of triglycerides,total cholesterol and low density lipoprotein,and enhancing the levels of high density lipoprotein in the serum/liver of mice.Pathological examination confirmed that ACT improved the organ structures of alcohol-damaged mice.The label free quantification proteomics analysis was used to assess the possible targets and pathways for ACT to play a protective role in mice with chronic alcohol-induced liver injury.Proteins associated with fatty acid metabolism,mitochondrial inner membrane and fibrinogen were selected based on the results of GO and Kyoto encyclopedia of genes and genomes(KEGG)analysis.The results of enzyme-linked immunosorbent assay(ELISA)assay revealed that ACT reversed the over-expression of the factors related fatty acid metabolism(stearoyl-CoA desaturase-1,STEAP family member 4 and carnitine acetyltransferase),mitochondrial inner membrane(leucine zipper/EF-handcontaining transmembrane protein 1,cytochrome P450 4A10,cytochrome P450 4A14 and cytochrome P450 8B1)and fibrinogen(fibrinogen like protein 1,fibrinogen gamma chain and fibrinogen beta chain)in liver of mice with chronic alcohol-induced liver injury.Furthermore,ACT regulated the phosphorylation of Akt and adenosine 5‘-monophosphate(AMP)-activated protein kinase(AMPK),which in turn improved peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1?);and downregulated the expression of cytochrome P450 2EI(CYP2E1)to suppress NF-?B signaling pathway.The study showed that ACT has at least partial hepatoprotective effects in mice with chronic alcohol-induced liver injury,and the mechanism is the modulation of the mitochondrial dysfunction and abnormal lipid metabolism,which may related to Akt/PGC-1? and CYP2E1.Therefore,this article expresses the protective effects of ACT on the mice with alcohol-induced liver injury.Our results provide valuable evidence to support the use of ACT as a medicine for ALD.