Alcoholic Liver Injury Protective Effects Of Antrodin A From Mycelium Of Antrodia Camphorata

Antrodia camphorata(A.camphorata)is a traditional folk anti-alcoholic and liverprotecting medicinal fungi in Taiwan.Its fruiting body has various activities,such as antitumor,anti-cancer and anti-inflammatory.Although A.camphorata fruit bodies have so many functions,their growth is extremely slow and natural yield is scarce,which hinders further research.Therefore,in recent years,many scholars took A.camphorata mycelium as the research object and found that it is rich in secondary metabolites,such as ubiquinones,maleic succinic acid derivatives,triterpenoids,polysaccharides,and so on.Literature survey found that A.camphorata mycelium can significantly inhibit the increase of ALT,AST,AKP and bilirubin in the serum of rats with acute alcoholism,and has a protective effect on the liver,but the mechanism of action is not clear.In this thesis,solid-state fermentation of A.camphorata mycelium was used as the research object.By constructing an alcohol-intervening L-O2 cell model and a mouse model of acute alcoholic liver injury,we explored the hepatoprotective activities and mechanisms of maleic/succinic acid derivatives and ubiquinones in A.camphorata mycelium.The main results obtained are as follows:(1)Four active substances,Antroquinonol B(Aq B),Antrodin C(Ad C),Antrodin A(Ad A),and Antroquinonol(Aq),were separated by silica gel column chromatography and analyzed by HPLC in the ethyl acetate layer extract(EALE)from mycelium of A.camphorata.The anti-alcoholic liver cell injury activity of these four active ingredients was screened.L-O2 cells in the model group(AL)contracted,became brighter and rounder,and some of the cells floated,resulting in a poor cell state and reduced viability.The situation of Aq B and Ad C groups was similar to that of AL group,with obvious changes in cell morphology and decreased adherence;only a small number of cells in Ad A and Aq groups showed morphological changes,and the cell status was better than that in AL group.In addition,Ad A and Aq can significantly reduce the activity of ALT and AST in the culture medium;although Aq B and Ad C have a tendency to decrease,there is no significant difference compared with the AL group.At the same time,Ad A and Aq significantly reduced the content of MDA in cells and increased the content of SOD and GSH.Comprehensive analysis showed that Ad A and Aq were better than Aq B and Ad C in resisting alcoholic liver cell damage and improving cell antioxidant capacity.(2)Ad A and Aq were used as the research objects to evaluate the hepatoprotective effect from the liver tissue pathology observation,liver effusion enzyme activity,liver lipid peroxidation and antioxidant enzyme activity in a mouse model of acute alcoholic liver injury.High doses of Ad A(Ad AH,200 mg/kg)and Aq(Aq H,200 mg/kg)reduced liver index,inhibited serum ALT,AST,and AKP levels,and reduced hepatocyte inflammation and lipid infiltration.Alcohol intake caused a significant increase in TG content in AL group,and the positive control group(PC)significantly reduced the serum TG level.The treatment group(ELALEH,Ad AL,Ad AH,Aq L,and Aq H)also performed similarly to the PC group,although there were no significant differences between the five groups.Compared with normal group(NC),serum HDL-C level of mice in AL group was significantly reduced.Except for the Ad AL group,the doses of ELAE,Ad A,Aq and the PC group can significantly increase serum HDL-C levels in mice.Comprehensive analysis showed that Ad AH and Aq H can reduce liver lipid peroxidation,increase liver antioxidant enzyme activity,and alleviate the damage caused by acute alcohol to the liver.Expression levels of genes associated with oxidative stress(CYP2e1,Nrf-2 and HO-1),lipid peroxidation(FAS),and immunomodulation(TNF-α and TLR4)in the liver were analyzed by RT-q PCR.Compared with the NC group,the m RNA expression levels of CYP2e1,FAS,TLR4 and TNF-α in the AL group were significantly increased,while the m RNA expression levels of Nrf-2 and HO-1 were significantly decreased.However,these changes were reversed by high doses of Ad A,as evidenced by the m RNA expressions of Nrf-2,HO-1,FAS,and TNF-α returned to normal levels,and the m RNA expressions of CYP2e1 and TLR4 were similar to those of the PC group.(3)The effects of Antrodia camphorata samples on the structure of intestinal flora in mice with acute alcohol liver injury were investigated.Alcohol intake can reduce the community diversity of intestinal flora in mice.The Shannon index and Simpson index show that high doses of EALE,Ad A and Aq H can prevent the reduction of community diversity.At the phylum level,the abundance of Firmicutes(33.38%)and Bacteroidetes(31.51%)in AL group decreased,and Proteobacteria(16.56%)increased.The community abundance of Firmicutes(37.75%,36.02% and 48.17%)increased and that of Proteobacteria(11.53%,8.42% and 10.22%)decreased after treatment with high doses of EALE,Ad A and Aq.Principal component analysis showed that the intestinal flora of mice in the AL group was different from that in the NC group.The sample points of EALEH group,Ad AH group and NC group were concentrated and clustered together,indicating that the intestinal flora species composition of these three groups of mice was similar.Although the Aq H group received preventive treatment,there was still a difference in species composition between the Aq H group and the NC group.The significance test of intergroup differences of intestinal bacteria in mice showed that alcohol treatment significantly changed the composition of intestinal flora,while supplementation with Ad A could mitigate dysbiosis of intestinal flora induced by alcohol and Faecalibaculum,Lactobacillus,and Coriobacteriaceae＿UCG-002 showed significant negative correlation with ALT,AST,AKP,and MDA.